Negatively charged minor groove binders

ABSTRACT

The present invention provides a negatively charged minor groove binding compound, oligonucleotide conjugates comprising the same, and methods for using the same. The negatively charged minor groove binding compounds of the present invention comprises an acidic moiety that is capable of being ionized under physiological conditions. In particular, the negatively charged minor groove binding compound of the present invention comprises a binding moiety that binds preferentially into a minor groove of a double, triple or higher stranded DNA, RNA, PNA or hybrids thereof. The binding moiety comprises at least one aryl moiety and an acidic moiety which is covalently attached to a phenyl portion of the aryl moiety or to a heteroatom of a heteroaryl portion of the aryl moiety.

CROSS REFERENCES TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.10/507,267, filed Apr. 6, 2005, which is the national stage ofApplication No. PCT/US03/07467, filed Mar. 11, 2003, which claims thebenefit of U.S. application Ser. No. 60/363,602, filed Mar. 11, 2002.The full disclosure of each of these filings is incorporated herein byreference for all purposes.

FIELD OF THE INVENTION

The present invention relates to a negatively charged minor groovebinder or salts thereof.

BACKGROUND OF THE INVENTION

Intercalating agents that bind to double stranded oligonucleotides(e.g., DNA, RNA or hybrids thereof) are well known in the art. Ingeneral, intercalating agents are aromatic molecules that non-covalentlybind to double stranded oligonucleotides by intercalating themselvesbetween interfacing purine and pyrimidine bases of the two strands ofdouble stranded oligonucleotides. Thus, oligonucleotides carrying, i.e.,connected to, an intercalating group can be used in a variety ofapplications, including as hybridization probes.

Similarly, minor groove binding agents are compounds that bindnon-covalently into the minor groove of a double strandedoligonucleotides. However, minor groove binding agents are generallyhigher molecular weight molecules than intercalating agents. Typically,the molecular weight of minor groove binder agents range from about 150to about 2000 Daltons. Minor groove binding agents generally bind in anon-intercalating manner into the minor groove of double strandedoligonucleotides. Thus, minor groove binding agents can be used forduplex stabilization and are useful as hybridization improving tools, aswell as other applications. For example, hybridization reagentscomprising a covalently attached oligonucleotides and minor groovebinders are described in U.S. Pat. No. 6,321,894. In addition, usingminor groove binders in sequence specific binding and meltingtemperature modulation have been described in U.S. Pat. Nos. 6,303,312and 6,221,589, respectively.

Minor groove binder agents usually have a chain of connected (conjugatedor non-conjugated) aromatic rings. The presence of a plurality ofaromatic rings renders the minor groove binders prone toself-association (aggregation) due to the pi-interaction between thearomatic systems. Aggregation reduces effective concentration of theminor groove binders (poorly soluble) and consequently their minorgroove binding performance. One method for reducing non-specificself-associations is to introduce an electrostatic charge within theminor groove binders. Some minor groove binding compounds bearingpositive charges at the terminal position of the molecules are known.See, for example, Reddy et al., Pharmacol. Therapeut., 1999, 84, 1-111.The positive charge improves the attraction to the negatively chargedDNA duplexes thereby increasing the DNA binding affinity. Unfortunately,however, the positive charge can also increase non-specific binding.

Therefore, there is a need for minor groove binding compounds withreduced self-associations and non-specific binding.

SUMMARY OF THE INVENTION

Some aspects of the present invention are based on a negatively chargedminor groove binding (NMGB) compound that binds preferentially into aminor groove of a double, triple or higher stranded oligonucleotides,e.g., DNA, RNA, PNA or hybrids thereof. Preferably, the negativelycharged minor groove binding compound binds to the oligonucleotideduplex in a non-intercalating manner.

In one aspect of the present invention, an oligonucleotide-negativelycharged minor groove binder conjugate is provided. The conjugatecomprises:

-   -   a negatively charged minor groove binder moiety comprising:        -   at least one aryl moiety, and        -   at least one acidic moiety capable of ionizing under            physiological conditions, wherein said acidic moiety is            covalently attached to at least one of said aryl moiety and            optionally comprises an acidic moiety linker; and    -   an oligonucleotide moiety which is covalently attached to said        negatively charged minor groove binder moiety.

The negatively charged minor groove binder moiety can be covalentlyattached to any portion of the oligonucleotide. In one embodiment, thenegatively charged minor groove binder moiety is attached to3′-position, 5′-position or an internal sugar moiety of theoligonucleotide. In another embodiment, the negatively charged minorgroove binder moiety is covalently attached to a heterocyclic baseportion of the oligonucleotide moiety.

The negatively charged minor groove binder moieties of the presentinvention comprise one or more acidic moieties. In one embodiment, thenegatively charged minor groove binder moiety comprises two or moreacidic moieties, preferably at least three acidic moieties.

Preferably, the aryl moiety of negatively charged minor groove binder isselected from the group consisting of phenyl, a heteroaryl, a fusedphenyl-heteroaryl, a fused heteroaryl-phenyl-heterocyclyl and acombination thereof.

In one embodiment, the acidic moiety is covalently attached to a phenylmoiety or a heteroatom of a heteroaryl portion of the aryl moiety,optionally through the acidic moiety linker. While any acidic moietywhich is capable of ionizing under physiological condition can be usedin the present invention, in one particular embodiment, the acidicmoiety has pKa of about 6 or less. Preferably, each of the acidic moietyis independently selected from the group consisting of:

-   -   (i) —(O)_(d)S(O)_(e)OH, wherein d is 0 or 1 and e is 1 or 2, and    -   (ii) —(O)_(f)P(O)_(g)(OR^(a1))_(h)(OH)_(i), wherein each R^(a1)        is independently selected from the group consisting of alkyl,        aralkyl and aryl; f is 0 or 1; each of g and h is independently        0, 1, or 2; and i is 1, 2 or 3, provided the sum of g+h+i is 2        or 3;    -   (iii) —CO₂H; and    -   (iv) salts thereof.        More preferably, each of the acidic moiety is independently        selected from the group consisting of —SO₂OH, —OPO₂(OH), —CO₂H,        and salts thereof.

In another embodiment, at least one of the acidic moiety is covalentlyattached to at least one of said aryl moiety through the acidic moietylinker. Preferably, the acidic moiety linker comprises a chain of atoms.Such chain of atoms can be arranged in a variety of manners such ascyclic, acyclic, aryl or a combination thereof of Preferably, thebackbone of the acidic moiety linker chain comprises from 1 to about 30atoms. Each of the backbone atom is independently selected from thegroup consisting of C, N, O, S, P. In addition, each of these backboneatoms can be substituted with appropriate substituents known to oneskilled in the art. For example, carbon atom can be substituted with ahydrogen, carbonyl oxygen, alkoxy, halide, amine, amide, cyano,hydroxyl. And sulfur and phosphorous atoms can be substituted with oneor more oxygen atoms. Preferably, carbon atoms of the chain backbone areindependently substituted with hydrogen or carbonyl oxygen.

In one particular embodiment, the acidic moiety linker is of theformula:—(X¹)_(a)—[C(═O)]_(b)—(R¹)_(c)—X²wherein

each of a, b and c is independently 0 or 1;

each X¹ is independently selected from the group consisting of:

-   -   (i) O,    -   (ii) NR², where each R² is independently selected from the group        consisting of hydrogen, alkyl, cycloalkyl and a nitrogen        protecting group, and    -   (iii) alkylene;

each R¹ is independently selected from the group consisting of alkylene,cycloalkylene, arylene and a combination thereof; and

each X² is independently said acid moiety.

The number of nucleotide units present in the oligonucleotide moiety canvary depending on a variety of factors. In one embodiment, theoligonucleotide moiety comprises from 3 to about 100 nucleotide units.However, it should be appreciated that the present invention is notlimited to this particular number of nucleotide units in theoligonucleotide moiety.

Still in another embodiment, the oligonucleotide-negatively chargedminor groove binder conjugate comprises a covalently attachedfluorophore moiety. The fluorophore moiety can be attached to anyportion of the conjugate, including the negatively charged minor groovebinder or a linker, if present. In addition, more than one fluorophoremoiety can be present in the conjugate. Preferably, the fluorophore iscovalently attached to the oligonucleotide moiety optionally via asecond linker moiety.

While any conventionally known fluorophores are suitable inoligonucleotide-negatively charged minor groove binder conjugates of thepresent invention, preferred fluorophores includes those with theemission wavelength of from about 400 to about 1000 nm.

In one embodiment, the negatively charged minor groove binder moiety iscovalently attached to the oligonucleotide moiety through a first linkermoiety. The first linker moiety can also comprise a covalently attachedquencher moiety. Preferably, the quencher moiety is compatible with thefluorophore moiety such that the emission wave of the fluorophore moietyis absorbed by the quencher moiety. In one embodiment, the absorbancemaximum of the quencher moiety is from about 400 nm to about 1000 nm.

In one specific embodiment of the present invention, theoligonucleotide-negatively charged minor groove binder conjugate is ofthe formula:

wherein

-   -   NMGB is said negatively charged minor groove binder;    -   ODN is said oligonucleotide;    -   FL is a fluorophore;    -   Q is a quencher;    -   L¹ is a first linker comprising a chain of from 3 to about 100        atoms selected from the group consisting of C, N, O, S, P and        combinations thereof;    -   L² is a second linker comprising a chain of from 1 to about 30        atoms selected from the group consisting of C, N, O, S, P and        combinations thereof; and    -   each of a₁, b₁, c₁ and d₁ is independently 0 or 1.

In one embodiment, the negatively charged minor groove binder moiety isof the formula:

wherein

-   -   n is an integer from 2 to 10;    -   R³ is selected from the group consisting of:        -   (a) alkoxy,        -   (b) aryloxy,        -   (c) R^(a)-O-L³-N(R^(b))-, where L³ is a third linker            comprising a chain of from 3 to 20 atoms selected from the            group consisting of C, N, O, S, P and combinations thereof;            and R^(a) is hydrogen, a hydroxyl protecting group or it is            attached to the first linker L¹; and R^(b) is hydrogen,            alkyl, cycloalkyl or a nitrogen protecting group,        -   (d) a moiety of the formula:

-   -   -   where            -   each of X³ is independently selected from the group                consisting of hydrogen, alkyl, alkoxy, halide, cyano,                nitro, said acidic moiety optionally comprising an                acidic linker and —NR^(b1)—C(═O)R^(c), where each R^(b1)                is hydrogen, alkyl, cycloalkyl or a nitrogen protecting                group, each R^(c) is independently selected from the                group consisting of hydrogen, alkyl, and cycloalkyl;

    -   each of R⁴ is independently selected from the group consisting        of:        -   (a) hydrogen,        -   (b) alkyl,        -   (c) the acidic moiety optionally comprising an acidic moiety            linker,        -   (d) —C(═O)—R⁷¹, where R⁷¹ is hydrogen, alkyl, hydroxy or            alkoxy,        -   (e) —NR′R″, where each of R′ and R″ is independently            hydrogen or alkyl, and        -   (f) -(alkylene)-OR⁷², where R⁷² is hydrogen or alkyl,

    -   each R⁵ is independently selected from the group consisting of:        -   (a) hydrogen,        -   (b) alkyl,        -   (c) alkoxy,        -   (d) cycloalkyl,        -   (e) halide,        -   (f) cyano,        -   (g) nitro,        -   (h) —[X⁴]_(m1)—C(═O)—[O]_(m2)—R⁸, wherein each of the            subscripts m1 and m2 is independently 0 or 1, X⁴ is O,            NR^(b1), where R^(b1) is hydrogen, alkyl, cycloalkyl or a            nitrogen protecting group and R⁸ is hydrogen, alkyl or            cycloalkyl, provided when m2 is 1, R⁸ is alkyl or            cycloalkyl, and        -   (i) —C(═O)—NR^(e)R^(f), where each of R^(e) and R^(f) is            independently hydrogen, alkyl, cycloalkyl and a nitrogen            protecting group,        -   (j) the acidic moiety optionally comprising an acidic moiety            linker,        -   (k) —NR′R″, where each of R′ and R″ is independently            hydrogen or alkyl, and        -   (l) -(alkylene)-OR⁷², where R⁷² is hydrogen or alkyl;

    -   each of R⁶ and R⁷ is selected from the group consisting of:        -   (a) hydrogen,        -   (b) alkyl,        -   (c) cycloalkyl,        -   (d) -L^(x)-Z^(x), where L^(x) is a linker comprising from 3            to 20 atoms selected from the group consisting of C, N, O,            S, P and combinations thereof; Z^(x) is hydrogen, a            protecting group, a solid support or a point of attachment            to said first linker L¹,        -   (e) the acidic moiety optionally comprising an acidic moiety            linker, and        -   (f) a moiety of the formula)            -(Z¹)_(j)-C(═O)—(R¹⁰)_(k)-[C(═O)]_(l)-R¹¹,        -   where            -   each of j, k and l is independently 0 or 1;            -   each Z¹ is independently selected from the group                consisting of O, NR¹² and alkylene;            -   each R¹⁰ is independently selected from the group                consisting of alkylene and cycloalkylene;            -   each R¹¹ is independently selected from the group                consisting of alkyl, alkoxy, aryloxy, —NR¹³R¹⁴,                —NR¹⁵—NR¹⁶R¹⁷, hydroxyalkyl and thioalkyl; and            -   each of R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷, is                independently selected from the group consisting of                hydrogen, alkyl, cycloalkyl and a nitrogen protecting                group;        -   (g) —NR′R″, where each of R′ and R″ is independently            hydrogen or alkyl;        -   (h) -(alkylene)-OR⁷², where R⁷² is hydrogen or alkyl; and        -   (i) —CHO,            provided at least one of X³, R⁴, R⁶, or R⁷ comprises said            acidic moiety optionally comprising said acidic moiety            linker, and provided that one of R³, R⁶, R⁷ and R^(a) is a            point of attachment to said first linker L¹. The negatively            charged minor groove binding (i.e., NMGB) moiety can be part            of or attached to R³ moiety and/or R⁶ moiety. Furthermore,            the NMGB can be attached to any portion of the sugar moiety,            e.g., the 3′- or 5′-position.

In another embodiment, the negatively charged minor groove binder moietyis of the formula:

wherein

-   -   W¹ is N or CR^(x30), where R^(x30) is hydrogen, alkyl, or        hydroxy, preferably R^(x30) is hydrogen, methyl, hydroxy or        trifluoromethyl;    -   W² is NR¹⁹, S or O;    -   p is an integer from 2 to 12;    -   each R¹⁹ is independently selected from the group consisting of:        -   (a) hydrogen,        -   (b) alkyl,        -   (c) a nitrogen protecting group, and        -   (b) said acidic moiety optionally comprising an acidic            moiety linker;    -   each of R¹⁸ and R²⁰ is independently selected from the group        consisting of:        -   (a) hydrogen,        -   (b) alkyl,        -   (c) cycloalkyl,        -   (d) said acidic moiety; and        -   (e) —(Z¹)_(j)-C(═O)—(R¹⁰)_(k)-[C(═O)]_(l)-R¹¹,        -   where j, k, l, Z¹, R¹⁰, R¹¹ are those define above;            provided at least one of R¹⁸, R¹⁹ or R²⁰ is said acidic            moiety, optionally comprising said acidic moiety linker, and            provided that one of R¹⁸ and R²⁰ is a point of attachment to            said first linker L¹. Preferably, the negatively charged            minor groove binder moiety is of the formula:

Yet in another embodiment, the negatively charged minor groove bindermoiety is of the formula:

wherein

R²¹ is an optionally substituted aryl-heterocyclyl;

each of R²² and R²⁴ is independently selected from the group consistingof hydrogen, alkyl, cycloalkyl and a nitrogen protecting group;

Ar¹ is optionally substituted aryl moiety; and

R²³ is selected from the group consisting of hydrogen and said acidicmoiety optionally comprising said acidic moiety linker, provided whenR²³ is hydrogen at least one of Ar¹ or R²¹ is substituted with saidacidic moiety optionally comprising said acidic moiety linker, andprovided that one of R²¹, R²², R²³ and R²⁴ is a point of attachment tosaid first linker L¹.

In one particular embodiment, the quencher moiety is a diazo moiety.Preferably, a diazo moiety of the formula:

wherein

-   -   Y is selected from the group consisting of substituted        phenyldiazenyl, nitro and —NR⁵⁰R⁵¹, where each of R⁵⁰ and R⁵¹ is        independently selected from the group consisting of hydrogen,        alkyl, cycloalkyl and a nitrogen protecting group;    -   each of z and w is independently an integer from 0 to 4;    -   each R^(z) is independently selected from the group consisting        of hydrogen, nitro, cyano, halide and —S(O)_(aa)NR⁵²R⁵³, where        aa is 0, 1 or 2 and each of R⁵² and R⁵³ is independently        selected from the group consisting of hydrogen, alkyl,        cycloalkyl and a nitrogen protecting group, or two adjacent        R^(z)'s and carbon atom to which they are attached to forms a        five- or six-membered ring having from zero to three heteroatoms        as ring members; and    -   each R^(w) is independently selected from the group consisting        of alkoxy, halide and —NR⁵⁴—C(═O)R⁵⁵, where R⁵⁴ is selected from        the group consisting of hydrogen, alkyl and a nitrogen        protecting group, and R⁵⁵ is selected from the group consisting        of hydrogen, alkyl and cycloalkyl, or two adjacent R^(w)'s and        carbon atom to which they are attached to forms a five- or        six-membered ring having from zero to three heteroatoms as ring        members.

In one particular embodiment, the quencher moiety is of the formula:

where Y, w, z, R^(w) and R^(z) are those defined herein.

In one embodiment, the quencher moiety is of the formula:

where w, z, R^(w) and R^(z) are those defined herein; and Y is selectedfrom the group consisting of nitro and —N(CH₃)₂.

In one particular embodiment, the fluorophore moiety, FL, is selectedfrom the group consisting of:

wherein

-   -   each of R⁶¹, R⁶², R⁶³, R⁶⁴, R⁶⁵, R⁶⁶ and R⁶⁷ is independently        selected from the group consisting of hydrogen, halide, nitro,        cyano, SO₃R⁷⁰, SO₂N(R⁷⁰)₂, C(O)O R⁷⁰, C(O)N(R⁷⁰)₂, CNS, —OR⁷⁰,        —OC(O)R⁷⁰, —SR⁷⁰, —CF₃, —NHC(O)R⁷⁰, —N(R⁷⁰)₂, wherein each R⁷⁰        is independently selected from the group consisting of hydrogen,        C₁-C₈ alkyl, aryl and a protecting group compatible with        oligonucleotide synthesis, or    -   two adjacent groups of R⁶¹ through R⁶⁶ together with the carbon        atoms to which they are attached form a five- or six-membered        ring having from zero to three heteroatoms as ring member; and    -   Z is O or S;        provided that at least one of R⁶¹ through R⁶⁷ is a point of        attachment to said second linker L² or to said oligonucleotide        ODN.

In one embodiment, the first linker, L¹, is selected from the groupconsisting of moieties the formula:

-   -   where a₂ and b₂ are independently an integer from 2 to 10;

where a₂, b₂ and c₂ are independently an integer from 2 to 10, X is O,CH₂ or NR^(i) and each of R^(g), R^(h) and R^(i) is independentlyhydrogen, alkyl, cycloalkyl or a nitrogen protecting group; and

where a₂, b₂ and c₂ are independently an integer from 2 to 10, X is O,CH₂ or NR^(i) and each of R^(g) and R^(i) is independently hydrogen,alkyl, cycloalkyl or a nitrogen protecting group.

Another aspect of the present invention provides a negatively chargedminor groove binding compound comprising a binding moiety that bindspreferentially into a minor groove of a double stranded oligonucleotide,wherein said binding moiety comprises:

-   -   at least one aryl moiety, and    -   at least one acidic moiety capable of ionizing under        physiological conditions, wherein said acidic moiety is        covalently attached to a phenyl moiety of said aryl moiety or a        heteroatom of a heteroaryl portion of said aryl moiety, wherein        said acidic moiety optionally comprises an acidic moiety linker.

In one embodiment, the aryl moiety is selected from the group consistingof phenyl, a heteroaryl, a fused phenyl-heteroaryl, a fusedheteroaryl-phenyl-heterocyclyl and a combination thereof. Preferably,each of the aryl moiety is independently selected from the groupconsisting of indole, benzofuran, pyrroloindole, hydropyrroloindole,phenyl, pyrrole, benzimidazole, imidazole, pyridine,6-phenylimidazo[4,5-b]pyridine, furan, thiazole and oxazole.

In one embodiment, the binding moiety comprises a plurality of arylmoieties. Preferably, at least three aryl moieties.

In one particular embodiment, the negatively charged minor groovebinding compound is of the formula:

wherein n, R³, R⁴, R⁵, R⁶ are those defined above, provided at least oneof X³, R⁴, R⁶ and R⁷ is an acidic moiety optionally comprising an acidicmoiety linker.

In another embodiment, the negatively charged minor groove bindingcompound is of the formula:

where p, R¹⁸, R²⁰, W¹ and W² are those defined herein, provided at leastone of R¹⁸, R¹⁹ (when W² is NR¹⁹) or R²⁰ is an acidic moiety, optionallycomprising an acidic moiety linker. In this embodiment, the negativelycharged minor groove binding compound is preferably of the formula:

where p, R¹⁸, R¹⁹ and R²⁰ are those defined herein, provided at leastone of R¹⁸, R¹⁹ or R²⁰ is an acidic moiety, optionally comprising anacidic moiety linker.

Yet in another embodiment, the negatively charged minor groove bindingcompound is of the formula:

where R²¹, R²², R²³, R²⁴ and Ar¹ are those defined herein, provided whenR²³ is hydrogen at least one of Ar¹ or R²¹ is substituted with an acidicmoiety, optionally comprising an acidic moiety linker. Preferably, R²²,R²⁴ and R²⁵ are hydrogen.

In one particular embodiment, R²¹ is selected from the group consistingof:

wherein

R²⁶ is selected from the group consisting of hydrogen, alkyl, and anitrogen protecting group;

R²⁷ is selected from the group consisting of:

-   -   (a) hydrogen,    -   (b) alkyl,    -   (c) cycloalkyl,    -   (d) said acidic moiety, optionally comprising said acidic moiety        linker,    -   (e) -(Z¹)_(j)-C(═O)—(R¹⁰)_(k)-[C(═O)]_(l)-R¹¹,    -   where        -   each of j, k and l is independently 0 or 1;        -   each Z¹ is independently selected from the group consisting            of O, NR¹² and alkylene;        -   each R¹⁰ is independently selected from the group consisting            of alkylene and cycloalkylene;        -   each R¹¹ is independently selected from the group consisting            of alkyl, alkoxy, aryloxy, —NR¹³R¹⁴, —NR¹⁵—NR¹⁶R¹⁷,            hydroxyalkyl and thioalkyl; and        -   each of R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ is independently            selected from the group consisting of hydrogen, alkyl,            cylcoalkyl, and a nitrogen protecting group, and    -   (f) -L^(x)Z^(x), where L^(x) is a linker comprising from 3 to 20        atoms selected from the group consisting of C, N, O, S, P and        combinations thereof, and Z^(x) is selected from the group        consisting of hydrogen, a protecting group or a solid support;        and

R²⁸ is selected from the group consisting of hydrogen and said acidicmoiety optionally comprising said acidic moiety linker.

In one embodiment, Ar¹ is selected from the group consisting of:

wherein

each of R²⁹, R³⁰ and R³² is independently selected from the groupconsisting of hydrogen and said acidic moiety, optionally comprisingsaid acidic moiety linker;

R³⁴ is selected from the group consisting of:

-   -   (a) hydrogen,    -   (b) alkyl,    -   (c) cycloalkyl,    -   (d) said acidic moiety, optionally comprising said acidic moiety        linker,    -   (e) -(Z¹)_(j)-C(═O)—(R¹⁰)_(k)-[C(═O)]_(l)-R¹¹,    -   where        -   each of j, k and l is independently 0 or 1;        -   each Z¹ is independently selected from the group consisting            of O, NR¹² and alkylene;        -   each R¹⁰ is independently selected from the group consisting            of alkylene and cycloalkylene;        -   each R¹¹ is independently selected from the group consisting            of alkyl, alkoxy, aryloxy, —NR¹³R¹⁴, —NR¹⁵—NR¹⁶R¹⁷,            hydroxyalkyl and thioalkyl; and        -   each of R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ is independently            selected from the group consisting of hydrogen, alkyl,            cylcoalkyl, and a nitrogen protecting group, and    -   (f) -L^(x)X^(x), where L^(x) is a linker comprising from 3 to 20        atoms selected from the group consisting of C, N, O, S, P and        combinations thereof, and Z^(x) is selected from the group        consisting of hydrogen, a protecting group or a solid support;

each of R³¹ and R³³ is independently selected from the group consistingof hydrogen, alkyl, a nitrogen protecting group and said acidic moietyoptionally comprising an acidic moiety linker;

each of R⁴⁰, R⁴³ and R⁴⁴ is independently selected from the groupconsisting of hydrogen, alkyl, and a moiety of the formula: -L^(x)Z^(x),-(Z¹)_(j)-C(═O)—(R¹⁰)_(k)-[C(═O)]_(l)-R¹¹, where L^(x), Z^(x), Z¹, j,R¹⁰, k, l, and R¹¹ are those defined herein.

each of R⁴¹ and R⁴² is independently selected from the group consistingof hydrogen, alkyl, and said acidic moiety which optionally comprisessaid acidic moiety linker; and

R⁴⁵ is selected from the group consisting of hydrogen, alkyl, alkoxy,cycloalkyl, halide, cyano, nitro, and a moiety of the formula:—[X⁴]_(m1)—C(═O)—[O]_(m2)—R⁸, where X⁴, m1, m2, and R⁸ are those definedherein.

In another embodiment, the negatively charged minor groove bindingcompound is covalently attached to a solid support, preferably through asolid support linker.

Another aspect of the present invention provides, a method foridentifying a nucleic acid comprising:

-   -   (a) incubating a first oligonucleotide with an oligonucleotide        probe; and    -   (b) identifying a hybridized nucleic acid;        wherein said oligonucleotide probe comprises:    -   a negatively charged minor groove binder moiety comprising:        -   at least one aryl moiety, and        -   at least one acidic moiety capable of ionizing under            physiological conditions, wherein said acidic moiety is            covalently attached to at least one of said aryl moiety,            optionally through an acidic moiety linker; and    -   an oligonucleotide moiety which is covalently attached to said        negatively charged minor groove binder moiety.

In one embodiment, the oligonucleotide probe is of the formula:

where NMGB, ODN, FL, Q, L¹, L² and k are those defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows structures of compounds that were used in a match andmismatch discrimination comparison test of Examples 4 and 5; and

FIG. 2 shows a calculated relative free energy difference, ΔΔG°(cal/mol), between match and mismatch (C/A) at positions 5, 6, 7, and 8in the conjugates at 50° C. of compounds of FIG. 1.

DEFINITIONS

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Throughout this disclosure, a squiggly line in a chemical structureindicates a point of attachment.

The terms “negatively charged minor groove binding compound”,“negatively charged minor groove binder” and “negative minor groovebinder” are used interchangeably herein and refer to a minor groovebinding compound comprising an acidic moiety that is capable of beingionized under physiological conditions. Preferably, the acidic moietyhas pKa of about 7 or less, more preferably pKa of about 6 or less,still more preferably pKa of about 5 or less, and most preferably pKa ofabout 4 or less.

The term “binds preferentially into a minor groove” means that themolecule binds within the double-stranded β-DNA minor groove in ahigh-affinity, preferably in a non-intercalative manner. Thus, a “minorgroove binder” refers to a molecule that is capable of binding withinthe minor groove of double-stranded DNA, double-stranded RNA, DNA-RNAhybrids, DNA-PNA hybrids, hybrids in which one strand is a PNA/DNAchimera and/or polymers containing purine and/or pyrimidine bases and/ortheir analogues which are capable of base-pairing to form duplex,triplex or higher order structures comprising a minor groove.

The terms “quencher” refers to an acceptor moiety in fluorescenceresonance energy transfer (FRET) detection method which is used in DNAor RNA probes.

The terms “fluorophore,” “fluorescent label” and “reporter” are usedinterchangeably herein and refer to a reporter moiety in fluorescenceresonance energy transfer (FRET) detection method which is used in DNAor RNA probes. Preferably, the fluorophore has a fluorescent emissionmaximum from about 400 to about 1000 nm, more preferably from about 400to about 900 nm, and still more preferably from about 400 to about 800nm. These compounds include, with their emission maxima in nm inbrackets, CY2™ (506), YO PRO™-1 (509), YOYO™-1 (509), Calcein (517),FITC (518) FLUORX™ (519), ALEXA™ (520), Rhodamine 110 (520),5-carboxyfluorescein (522), OREGON GREEN™ 500 (522), OREGON GREEN™ 488(524), RIBOGREEN™ (525), RHODAMINE GREEN™ (527), Rhodamine 123 (529),MAGNESIUM GREEN™ (531), CALCIUM GREEN™ (533), TO-PRO™ -1 (533), TOTO®-1(533), JOE (548), BODIPY 530/550 (550), Dil (565), BODIPY® (568), BODIPY558/568 (568), BODIPY 564/570 (570), CY3 ™ (570), ALEXA™ 546 (570),TRITC (572), MAGNESIUM ORANGE™ (575), Phycoerythrin R&B (575), RhodaminePhalloidin (575), CALCIUM ORANGE™ (576), Pyronin Y (580), Rhodamine B(580), tetramethylrhodamine (582), Rhodamine RED™ (590), CY3.5™ (596),ROX (608), Calcium CRIMSON™ (615), ALEXA™ 594 (615), TEXAS RED (615),NILE RED (628), YO 3 (631), YOYO™-3 (631), R-phycocyanin (642),C-Phycocyanin (648), TO PRO™-3 (660), TOTO (D-3 (660), DiD DilC(5)(665), CY5™ (670), Thiadicarbocyanine (671), Cy5.5 (694).

The terms “oligonucleotide,” “nucleic acid” and “polynucleotide” areused interchangeable herein. These terms refer to a compound comprisingnucleic acid, nucleotide, or its polymer in either single- ordouble-stranded form, e.g., DNA, RNA, analogs of natural nucleotidesdescribed below, and hybrids thereof. Unless otherwise limited, theterms encompass known analogs of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally-occurring nucleotides.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). A“subsequence” or “segment” refers to a sequence of nucleotides thatcomprise a part of a longer sequence of nucleotides.

The terms “linker,” “linking group” and “linking chain” are usedinterchangeably herein and refer to a chain of atoms that is used toassemble various portions of the molecule or to covalently attach themolecule (or portions thereof) to a solid support. Typically a linkerhas functional groups that are used to interact with and form covalentbonds with functional groups in the ligands or components (e.g.,fluorophores, oligonucleotides, negatively charged minor groove binders,or quenchers) of the conjugates described and used herein. Examples offunctional groups on the linking groups (prior to interaction with othercomponents) include —NH2, —NHNH2, —ONH2, —NH—C═(O)—NHNH2, —OH, and —SH.A linker can include acyclic portions, cyclic portions, aromatic ringsor combinations thereof. Preferably, the linker is cyclic, acyclic or acombination thereof. Typically, the linker (i.e., backbone) comprises aprescribed number of atoms selected from the group consisting of C, N,O, S, P and a combination thereof. Each of the atoms can be substitutedwith appropriate substituents known to one skilled in the art. Forexample, carbon atom can be substituted with a hydrogen, carbonyloxygen, alkoxy, halide, amine, amide, cyano, hydroxyl. And sulfur andphosphorous atoms can be substituted with one or more oxygen atoms.

“Alkyl” means a linear or branched saturated monovalent hydrocarbonmoiety which can be optionally substituted with one or more halides.Exemplary alkyl groups include, but are not limited to, methyl,trifluoromethyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl, and thelike.

“Alkylene” means a linear saturated divalent hydrocarbon moiety of oneto ten, preferably one to six, carbon atoms or a branched saturateddivalent hydrocarbon moiety of three to ten, preferably three to six,carbon atoms, e.g., methylene, ethylene, propylene, 2-methylpropylene,pentylene, and the like.

“Alkoxy” refers to a moiety of the formula —ORa2, where Ra2 is alkyl asdefined herein.

“Aryl” means any monocyclic, bicyclic, tricyclic or tetracyclic ringmoiety comprising a phenyl moiety and/or a heteroaryl moiety, which canoptionally be fused to a heterocyclyl moiety. With the understandingthat no two phenyl moieties are bonded together, i.e., no naphthylmoiety is present on the aryl group. With further understanding that theattachment of the aryl group is through a carbon or a heteroatom atom ofa phenyl or a heteroaryl moiety. Moreover, the aryl group can beoptionally substituted with one or more substituents selected from thegroup consisting of alkyl, heteroalkyl, heterocyclyl, halo, nitro,nitroso, cyano, carboxy and acyl. Exemplary aryl moieties include, butare not limited to, phenyl, fused phenyl-heteroaryl, and fusedheteroaryl-phenyl-heterocyclyl as defined herein. Specific examples ofaryl moieties include, but are not limited to, phenyl, indole,pyrroloindole, hydropyrroloindole, benzimidazole, imidazole, pyridine,6-phenylimidazo[4,5-b]pyridine and furan.

“Aryloxy” refers to a moiety of the formula —ORa3, where Ra3 is an arylmoiety as defined herein.

“Cycloalkyl” refers to a saturated monovalent cyclic hydrocarbon moietyof three to seven ring carbons. The cycloalkyl can be optionallysubstituted independently with one or more substituents.

“Heteroaryl” means a monovalent monocyclic aromatic moiety of 5 to12,preferably 5 or 6, ring atoms containing one, two, or three ringheteroatoms selected from the group consisting of N, O, or S, theremaining ring atoms being C. The heteroaryl ring can be optionallysubstituted independently with one or more substituents selected fromthe group consisting of alkyl, heteroalkyl, heterocyclyl, halo, nitro,nitroso, cyano, carboxy, acyl. More specifically the term heteroarylincludes, but is not limited to, pyridyl, furanyl, thiophenyl,thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrrolyl,pyrazolyl, pyrimidinyl and the derivatives thereof.

“Fused phenyl-heteroaryl” means a bicyclic ring moiety comprising aphenyl moiety and a heteroaryl moiety as defined herein in which thephenyl moiety and the heteroaryl moiety share two or more, preferablytwo, common atoms. Such bicyclic ring moiety can be represented by ageneral formula:

where Het¹ is heteroaryl and Ph is phenyl.

“Fused heteroaryl-phenyl-heterocycloalkyl” means a tricyclic ring moietycomprising a phenyl moiety as the core which is covalently attached toboth a heteroaryl moiety and a heterocyclyl moiety as defined herein inwhich the phenyl moiety independently shares at least two or more,preferably two, common atoms with the heteroaryl and the heterocyclylmoieties. Such tricyclic ring moiety can be represented by a generalformula:

where Het¹ is heteroaryl, Ph is phenyl and Het² is heterocyclyl.

“Fused heteroaryl-heterocyclyl” refers to bycyclic, tricyclic ortetracyclic ring moiety comprising a heterocyclyl moiety and aheteroaryl moiety as defined herein in which the heterocyclyl moiety andthe heteroaryl moiety share two or more, preferably two, common atomswith the understanding that the attachment is through the heteroatom ofthe heterocyclyl moiety. Such ring moiety can be represented by ageneral formula:

where Het¹ is heteroaryl and Hetc is heterocyclyl with X being aheteroatom of the heterocyclyl moiety.

“Fused aryl-heterocyclyl” means any bicyclic, tricyclic or tetracyclicring moiety comprising a phenyl moiety and/or a heteroaryl moiety, whichis fused to a heterocyclyl moiety. With the understanding that theattachment of the fused aryl-heterocyclyl group is through a heteroatomof a heterocyclyl moiety. Specific examples of fused aryl-heterocyclylmoieties include, but are not limited to,1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (i.e., CDPI), CPI,pyrroloindole and hydropyrroloindole.

“Heterocycloalkyl” refers to a saturated monocyclic or bicyclic moietyof 3 to 12, preferably 5 or 8, ring atoms in which one or two ring atomsare heteroatoms selected from N, O, or S(O)_(n) (where n is an integerfrom 0 to 2), the remaining ring atoms being C, where one or two C atomscan optionally be replaced by a carbonyl group. The heterocyclyl ringcan optionally be substituted with one or more substituents with theunderstanding that the heterocyclyl moiety is attached through a carbonatom of the heterocycloalkyl moiety.

“Heterocyclyl” refers to a non-aromatic monocyclic or bicyclic moiety of3 to 12, preferably 5 or 8, ring atoms in which one or two ring atomsare heteroatoms selected from N, O, or S(O)_(n) (where n is an integerfrom 0 to 2), the remaining ring atoms being C, where one or two C atomscan optionally be replaced by a carbonyl group. The heterocyclyl ringcan optionally be substituted with one or more substituents with theunderstanding that the heterocyclyl moiety is attached through aheteroatom.

The terms “CDPI_(n)” and “DPI_(n)” are used interchangeably herein andrefer to a moiety of the general formula:

where X, R⁴ and R⁵ are those defined herein. Formula Z¹-CDPI_(n)-Z²means there are n-mer of CDPI (or DPI) with Z¹ being attached to theterminal carbonyl carbon and Z² being attached to the nitrogen atom ofthe pyrrole ring system. For example, a compound of the formula:

can be represented by a short hand notation PhO-CDPI₂-C(═O)—NH₂.

“Protecting group” refers to a moiety, except alkyl or cycloalkyl group,that when attached to a reactive group in a molecule masks, reduces orprevents that reactivity. Examples of protecting groups can be found inT. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons, New York, 1999, Harrison and Harrisonet al., Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wileyand Sons, 1971-1996), and “Protection of Nucleosides for OligonucleotideSynthesis,” Current Protocols in Nucleic Acid Chemistry, ed. by Boyle,A. L., John Wiley & Sons, Inc., 2000, New York, N.Y., all of which areincorporated herein by reference in their entirety. Representativehydroxy protecting groups include acyl groups, benzyl and trityl ethers,tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.Representative amino protecting groups include, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl(Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES),trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC),and the like.

“Corresponding protecting group” means an appropriate protecting groupcorresponding to the heteroatom to which it is attached.

“Nitrogen protecting group” means an appropriate protecting groupcorresponding to the nitrogen atom to which the protecting group isattached. Such nitrogen atom can be in the form of an amine, an amide, aurea, an imine or other nitrogen functional group known to one skilledin the art. Suitable nitrogen protecting group for each nitrogen atomfunctional group are well known to one skilled in the art. See, forexample, T. W. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 3^(rd) edition, John Wiley & Sons, New York, 1999, Harrisonand Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8(John Wiley and Sons, 1971-1996), and “Protection of Nucleosides forOligonucleotide Synthesis,” Current Protocols in Nucleic Acid Chemistry,ed. by Boyle, A. L., John Wiley & Sons, Inc., 2000, New York, N.Y., allof which were incorporated by reference above.

“Leaving group” has the meaning conventionally associated with it insynthetic organic chemistry, i.e., an atom or a group capable of beingdisplaced by a nucleophile and includes halo (such as chloro, bromo, andiodo), alkanesulfonyloxy, arenesulfonyloxy, alkylcarbonyloxy (e.g.,acetoxy), arylcarbonyloxy, mesyloxy, tosyloxy,trifluoromethanesulfonyloxy, aryloxy (e.g., 2,4-dinitrophenoxy),methoxy, N,O-dimethylhydroxylamino, and the like.

“Pharmaceutically acceptable excipient” means one or more excipientsthat are useful in preparing a pharmaceutical composition. Excipientsare generally safe, non-toxic and neither biologically nor otherwiseundesirable, and include excipients that are acceptable for veterinaryuse as well as human pharmaceutical use.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include:salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.]

The terms “pro-drug” and “prodrug” are used interchangeably herein andrefer to any compound which releases an active parent drug according toFormula I in vivo when such prodrug is administered to a mammaliansubject. Prodrugs of a compound of Formula I are prepared by modifyingone or more functional group(s) present in the compound of Formula I insuch a way that the modification(s) may be cleaved in vivo to releasethe parent compound. Prodrugs include compounds of Formula I wherein ahydroxy, amino, or sulfhydryl group in a compound of Formula I is bondedto any group that may be cleaved in vivo to regenerate the freehydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugsinclude, but are not limited to, esters (e.g., acetate, formate, andbenzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) ofhydroxy functional groups in compounds of Formula I, and the like.

“Treating” or “treatment” of a disease includes: (1) preventing thedisease, i.e., causing the clinical symptoms of the disease not todevelop in a mammal that may be exposed to or predisposed to the diseasebut does not yet experience or display symptoms of the disease; (2)inhibiting the disease, i.e., arresting or reducing the development ofthe disease or its clinical symptoms; or (3) relieving the disease,i.e., causing regression of the disease or its clinical symptoms.

“A therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

The term “treating”, “contacting” or “reacting” when referring to achemical reaction means to add or mix two or more reagents underappropriate conditions to produce the indicated and/or the desiredproduct. It should be appreciated that the reaction which produces theindicated and/or the desired product may not necessarily result directlyfrom the combination of two reagents which were initially added, i.e.,there may be one or more intermediates which are produced in the mixturewhich ultimately leads to the formation of the indicated and/or thedesired product.

As used herein, the terms “those defined above” and “those definedherein” when referring to a variable incorporates by reference the broaddefinition of the variable as well as preferred, more preferred and mostpreferred definitions, if any. Such variables refer to variables thatare not part of a schematic illustrations.

DETAILED DESCRIPTION OF THE INVENTION

The presence of a plurality of aromatic rings in minor groove bindersmakes them prone to self-association due to the pi-interaction betweenthe aromatic systems. One can reduce non-specific self-associations byintroducing an electrostatic charge within the minor groove binders.However, because oligonucleotides are negatively charged, it is expectedthat introduction of one or more negative charges to a minor groovebinder will result in electrostatic repulsion between the minor groovebinder and DNA or RNA. This electrostatic repulsion, in turn, isexpected to reduce the binding affinity of the minor groove binder tooligonucleotides. Thus, conventional electrostatic charged minor groovebinding compounds contain a positive charge at the terminal position ofthe molecules. In addition to reducing aggregation, the positive chargealso improves the affinity of the positively charged minor groovebinders to negatively charged DNA duplexes. Unfortunately, the positivecharge also increases non-specific binding.

I. Overview

The present invention is based on a surprising and unexpected discoveryby the present inventors that negatively charged minor groove bindersretained good oligonucleotide binding affinities while significantlyreducing or eliminating the aggregation problems associated with neutralminor groove binders.

The negative minor groove binders of the present invention are useful ina variety of application including hydridization assays. While thenegative minor groove binder can be used alone, it is particularlyuseful when covalently bound to an oligonucleotide, a quencher, afluorophore, or a combination thereof.

II. Negatively Charged Minor Groove Binders

The negative minor groove binders of the present invention comprise atleast one binding moiety, and at least one acidic moiety which iscapable of ionizing under physiological conditions. As such, the acidicmoiety has pKa of about 7 or less. Preferably pKa of about 6 or less,more preferably pKa of about 5 or less and still more preferably pKa ofabout 4 or less. Exemplary acidic moieties which are capable of ionizingunder physiological conditions include moieties of the formula—(O)_(a)S(O)_(b)OH, wherein a is 0 or 1 and b is 1 or 2,—(O)_(c)P(O)_(d)(OR^(a1))_(e)(OH)_(f), wherein each R^(a1) isindependently selected from the group consisting of alkyl, aralkyl andaryl; c is 0 or 1; each of d and e is independently 0, 1, or 2, and f is1, 2 or 3, provided the sum of d+e+f is 2 or 3; —CO₂H; and saltsthereof. Preferably, each of the acidic moiety in negative minor groovebinder is independently selected from the group consisting of —SO₂OH,OPO₂(OH), —CO₂H, and salts thereof. As used herein, the term “saltsthereof” refers to alkaline metal salts, alkaline-earth metal salts,transition metal salts, ammonium salts, and alkyl substituted ammoniumsalts. Preferred salts include sodium, potassium, lithium, calcium, andmagnesium.

The binding moiety comprises at least one aryl moiety. Preferably, theacidic moiety is covalently attached, optionally via an acidic moietylinker, to a phenyl portion or a heteroatom of a heteroaryl portion ofthe aryl moiety. In one embodiment, the binding moiety is selected fromthe group consisting of (a) a phenyl moiety; (b) a heteroaryl moiety;(c) a fused phenyl-heteroaryl moiety; (d) a fusedheteroaryl-phenyl-heterocyclyl moiety; or (e) a combination thereof Eachof which can optionally be substituted with one or more conventionalsubstituents known to one skilled in the art. Preferably, the bindingmoiety is selected from the group consisting of (a) a heteroaryl moiety;(b) a fused aryl-heteroaryl moiety; (c) a fusedheteroaryl-aryl-heterocyclyl moiety; and (d) a combination thereof

In one embodiment, the binding moiety comprises a plurality of arylmoieties. Preferably at least three aryl moieties, and more preferablyat least five aryl moieties. The aryl moieties can be linked directed toeach other or can be linked through an aryl moiety linker comprisingfrom 1 to about 10 atoms in the linking chain. In one preferredembodiment, the aryl moieties are covalently linked by an amide linkage(e.g., Ar^(x)—R^(q1)—C(═O)—NR^(q2)—R^(q1)—Ar^(y)), a urea linkage (e.g.,Ar^(x)—R^(q1)—NR^(q2)—C(═O)—NR^(q2)—R^(q1)—Ar^(y)) or combinationsthereof, where each of Ar^(x) and Ar^(y) is independently an aryl group,each of R^(q1) is independently a bond or a linker comprising a chain of1 to about 8 atoms, and each of R^(q2) is independently hydrogen, alkyl,cycloalkyl or a nitrogen protecting group. Preferably, each of R^(q1) isindependently C₁-C₈ alkylene.

In one particular embodiment, each of the aryl moiety is independentlyselected from the group consisting of indole, benzofuran, pyrroloindole,hydropyrroloindole, phenyl, pyrrole, benzimidazole, imidazole, pyridine,6-phenylimidazo[4,5-b]pyridine, furan, thioazole and oxazole.

The negative minor groove binders of the present invention comprise asufficient amount of acidic moieties to provide a significant reductionin aggregation, and provides a sufficient binding affinity tooligonucleotides to be useful. In one particular embodiment, thenegatively charged minor groove binder comprises a plurality of acidicmoieties. The acidic moieties can be attached to the aryl groups in avariety of combinations. For example, each acidic moiety can be attachedto a separate aryl group or some aryl groups can have one or more acidicgroups and some aryl groups can have no acidic group attached. Stillalternatively, all the acidic moieties can be attached to a single arylmoiety.

Preferably, the negatively charged minor groove binder comprises atleast two acidic moieties, more preferably at least about 3 acidicmoieties, and still more preferably at least about 5 acidic moieties.However, it should be appreciated that the actual number of acidicmoieties are not limited to these specific quantities and examples givenherein. The actual number of acidic moieties present in the negativeminor groove binder is ultimately determined by the above describedrequirement, i.e., it should provide a significant reduction inaggregation while retaining a sufficient binding affinity tooligonucleotides. Thus, the number of acidic moieties in the negativeminor groove binder can vary significantly depending on a variety offactors, including the overall structure of the negative minor groovebinder, and the presence of other moieties, such as an oligonucleotide,a quencher and/or a fluorophore.

The acidic moiety is covalently attached to at least one of the arylmoiety. The acidic moiety can be directly linked to the aryl moiety oroptionally via an acidic moiety linker. The acidic moiety linkercomprises a chain of atoms arranged in a cyclic and/or acyclic manner.Typically, this linking chain comprises from 1 to about 30 atoms. Insome embodiments, carbon atoms are substituted with hydrogen or carbonyloxygen.

In one particular embodiment, the combination of acidic moiety andacidic moiety linker is of the formula:—(X¹)_(a)—[C(═O)]_(b)—(R¹)_(c)—X²where X¹, X², a, b, c and R¹ are those defined herein. Preferably whenX¹ is alkylene, it is C₁-C₆ alkylene.

One particularly useful negatively charged minor groove binding compoundof the present invention is of the formula:

where n, R³, R⁴, R⁵, R⁶ and X³ are those defined herein, provided atleast one of X³, R⁴, R⁶ and R⁷ is an acidic moiety optionally comprisingan acidic moiety linker. Preferably n is 2 to 8.

In another embodiment, the negatively charged minor groove bindingcompound of the present invention is of the formula:

where p, W¹, W², R¹⁸ and R²⁰ are those defined herein, provided at leastone of R¹⁸, R¹⁹ (when W² is NR¹⁹) and R²⁰ is an acidic moiety,optionally comprising an acidic moiety linker. In this embodiment, thenegatively charged minor groove binding compound is preferably of theformula:

where p, R¹⁸, R¹⁹ and R²⁰ are those defined herein.

In yet another embodiment of the present invention, the negativelycharged minor groove binding compound is of the formula:

where Ar¹, R²¹, R²², R²³ and R²⁴ are those defined herein.

Preferably, R²² and R²⁴ of compound of Formula III are hydrogen.

Preferably, R²¹ is a moiety of the formula:

wherein

R²⁶ is selected from the group consisting of hydrogen, alkyl, and anitrogen protecting group;

R²⁷ is selected from the group consisting of:

-   -   (a) hydrogen,    -   (b) alkyl,    -   (c) cycloalkyl,    -   (d) said acidic moiety, optionally comprising said acidic moiety        linker,    -   (e) -(Z¹)_(j)-C(═O)—(R¹⁰)_(k)-[C(═O)]_(l)-R¹¹,    -   where j, k, l, Z¹, R¹⁰ and R¹¹ are those defined herein;    -   (f) -L^(x)Z^(x), where L^(x) and Z^(x) are those defined herein;        and

R²⁸ is selected from the group consisting of hydrogen and said acidicmoiety optionally comprising said acidic moiety linker.

Preferably, Ar¹ of compounds of Formula III is selected from the groupconsisting of:

wherein

each of R²⁹, R³⁰ and R³² is independently selected from the groupconsisting of hydrogen and said acidic moiety, optionally comprisingsaid acidic moiety linker;

R³⁴ is selected from the group consisting of:

-   -   (a) hydrogen,    -   (b) alkyl,    -   (c) cycloalkyl,    -   (d) said acidic moiety, optionally comprising said acidic moiety        linker,    -   (e) -(Z¹)_(j) 13 C(═O)—(R¹⁰)_(k)—[C(═O)]_(l)—R¹¹,    -   where j, k, l, Z¹, R¹⁰ and R¹¹ are those defined herein;    -   (f) -L^(x)Z^(x), where L^(x) and Z^(x) are those defined herein;

each of R³¹ and R³³ is independently selected from the group consistingof hydrogen, alkyl, a nitrogen protecting group and said acidic moietyoptionally comprising an acidic moiety linker;

each of R⁴⁰, R⁴³ and R⁴⁴ is independently selected from the groupconsisting of hydrogen, alkyl, and a moiety of the formula: -L^(x)Z^(x),—(Z¹)_(j)—C(═O)—(R¹⁰)_(k)—[C(═O)]_(l)—R¹¹, where L^(x), Z^(x), Z¹, j,R¹⁰, k, l, and R¹¹ are those defined herein.

each of R⁴¹ and R⁴² is independently selected from the group consistingof hydrogen, alkyl, and said acidic moiety which optionally comprisessaid acidic moiety linker; and

R⁴⁵ is selected from the group consisting of hydrogen, alkyl, alkoxy,cycloalkyl, halide, cyano, nitro, and a moiety of the formula:—[X⁴]_(m1)—C(═O)—[O]_(m2)—R⁸, where X⁴, m1, m2, and R⁸ are those definedherein.

A particularly useful negatively charged minor groove binding compoundsof Formula III include those in which R²¹ is a moiety of Formula IV andAr¹ is a moiety of Formula V, VI, VII, VIII, or IX.

The negatively charged minor groove binding compounds of the presentinvention can also be covalently attached to a solid support. Suchcovalent attachment can be a direct linkage or, preferably through asolid support linker comprising 1 to about 30 atoms in the linkingchain.

Synthesis of Negatively Charged Minor Groove Binders

Negatively charged minor groove binding compounds of the presentinvention can be synthesized using a variety of methods. One particularmethod for synthesizing compounds of Formula I comprising CDPI moiety isillustrated in Scheme I below.

As the above synthetic Scheme I shows, PFPO-CDPI_(m)-C(═O)—NH₂ (1-5)(where m=n+2 and PFPO is pentafluorophenoxide, i.e., pentafluorophenoxy,moiety), can be prepared by selective deprotection of tert-butylcarbamate and coupling the deprotected amine group with a suitablysubstituted pyrroloindoline 1-2 or 1-2a. Selective deprotection oftert-butyl carbamate group can be achieved by any of the conventionalmethods known to one skilled in the art including deprotecting reagentsand conditions described in the above incorporated Protective Groups inOrganic Synthesis, 3^(rd) edition, and Compendium of Synthetic OrganicMethods, Vols. 1-8.

Typically, the tert-butyl carbamate is selectively removed by reactingwith an acid, such as trifluoroacetic acid (i.e., TFA). The deprotectioncan be carried out using TFA as both a solvent and a deprotectingreagent. In some cases, the deprotection is carried out using a solutionof TFA in an organic solvent, such as methylene chloride, chloroform andother inert organic solvent. The reaction temperature is typically roomtemperature. However, lower or higher reaction temperature can also beused depending on the nature of the starting material. For example,reaction temperature of about 0° C. provides slower reaction but higherselectivity, while reaction temperature at 30° C. or higher typicallyresults in shorter reaction time but may yield slightly lowerselectivity and/or product yield. The reaction time can range from fewminutes to few hours depending on a variety of conditions such as theconcentration of the starting material, reaction temperature and whetherTFA is used as both a solvent and a deprotecting reagent or whether aninert organic solvent is used. Typically, the selective deprotection ofthe tert-butyl carbamate can be achieved within about 1 hr.

After the selective deprotection of the tert-butyl carbamate group, theresulting free amine is reacted with pyrroloindoline 1-2 or 1-2a toprovide an oligomer of CDPI. The difference between pyrroloindolines 1-2and 1-2a is the presence or the absence of the acidic moiety,respectively. Therefore, covalent attachment of pyrroloindoline 1-2 or1-2a depends on whether an acidic group on the phenyl portion of thepyrroloindoline is desired or not. The coupling reaction results incovalent attachment of the free amine to the ester carbonyl groupselectively. A carbonyl carbon of an ester group is generally morereactive than a carbonyl carbon of a carbamate group. This difference inthe reactivity is increased even further by the presence of apentafluorophenyl (i.e., PFP) group on the ester group. Thus, thecoupling reaction of the present invention results in almost exclusivecovalent attachment of the free amine to the ester carbonyl carbon atom.While pyrroloindolines 1-2 and 1-2a are depicted with PFP group, it isintended that such pyrroloindolines with other carbonyl activating groupare also within the scope of the present invention. As used herein“carbonyl activating group” refers to a moiety which increases thereactivity of the carbonyl carbon of an ester relative to a similaralkyl ester group (e.g., ethyl).

Suitable coupling reaction conditions between an ester group and anamine group are well known to one skilled in the art. Typically, thecoupling reaction involves suspending or, preferably dissolving, thefree amine in an inert relatively polar organic solvent, such as DMF,DMSO, DME, and the like, and adding pyrroloindoline 1-2 or 1-2a in thepresence of a non-nucleophilic base, such as tertiary amine compounds,bicarbonates and carbonates. The reaction is conveniently carried out atroom temperature and is complete within few hours. Often the resultingcoupled product precipitates out of the reaction mixture making theproduct recovery convenient. Typically, the product is simply filtered,washed and dried under vacuum.

As can be seen in Scheme I, the pyrroloindoline 1-2 and 1-2a contain atert-butyl carbamate group. Therefore, the resulting coupled product canbe subjected to selective deprotection and covalent attachment withanother pyrroloindolines 1-2 and 1-2a. This process of selectivedeprotection and coupling is repeated until n number of pyrroloindolinemoieties are coupled to the starting material. At least one of thecoupling of a selectively deprotected tert-buty carbamate involvespyrroloindoline 1-2, such that the resulting CDPI_(m) derivative 1-5comprises an acidic moiety of the present invention. After the finaltert-butyl carbamate group is deprotected, the resulting free amine iscoupled with (i.e., covalently attached to) an urea derivative ofpyrroloindoline 1-3. Pyrroloindoline 1-3 comprises tetrafluorophenylester moiety as a carbonyl activating group. However, similar topyrroloindolines 1-2 and 1-2a discussed above, the ester moiety ofpyrroloindoline 1-3 can also be activated with other conventionalcarbonyl activating group known to one skilled in the art.

The p-nitrophenylethyl ester moiety of Compound 1-4 is then selectivelyremoved and trans-esterified with pentafluorophenyl-trifluoroacetate(i.e., PFP-TFA) to provide PFPO-CDPI_(m)-C(═O)—NH₂ (1-5). Selectiveremoval of the p-nitrophenylethyl ester moiety of Compound 1-4 can beachieved using a sterically hindered base such as DBU. Typically, amixture of Compound 1-4 and DBU in an inert relatively polar organicsolvent, such as DMF, DME and DMSO, is heated to about 50° C. for about40 min. to provide a salt of the carboxylic acid. This salt is filtered,washed, dried and suspended in anhydrous organic solvent, e.g., DMF, inthe presence of a non-nucleophilic base, such as tertiary amine.Addition of pentafluorophenyl trifluoroacetate then results intrans-esterification reaction to provide PFPO-CDPI_(m)-C(═O)—NH₂ (1-5).Compound 1-5 contains activated ester group, and therefore it can becovalently attached to a variety of compounds, including,oligonucleotides, such as DNA, RNA, PNA, hybrids thereof; fluorescentcompounds; quencher compounds; and other suitable compounds.

Compound 1-2 can be produced by a variety of methods. In one particularembodiment, Compound 1-2 is synthesized from indoline sulfonic acid asshown in Scheme II below.

The starting material in Scheme II, indoline-6-sulfonic acid 2-1, can beprepared by a known procedure including the method disclosed in U.S.Pat. No. 4,405,788. Protection of the free amino group of indoline ofindoline-6-sulfonic acid 2-1 followed by nitration of the phenyl ringprovides a nitro indoline 2-3. While Scheme II illustrates the use ofacetyl protecting group for the amino group, it should be appreciatedthat any conventional amino protecting group that is suitable (i.e.,none interfering) for subsequent reaction conditions can be used.Nitration of the phenyl ring moiety of the indoline is well known in theart and can be conveniently achieved at 0° C. by using fuming nitricacid in sulfuric acid solution.

The nitro group is then reduced and converted to a diazonium salt 2-5.Treatment of the diazonium salt 2-5 with ethyl pyruvate in the presenceof a Lewis acid affords aza compound 2-6, which is then cyclized toprovide pyrroloindoline 2-7. The cyclization of the aza compound 2-6 isachieved using an acid catalyst. Typically refluxing conditions usingtrifluoroacetic acid is employed for cyclization reaction. To provide adicarbonyl CDPI compound 1-2, the acetyl amino protecting group ofpyrroloindoline 2-7 is then replaced with a Boc protecting group, andthe free carboxylic acid is esterified as a pentafluorophenoxy ester.Compound 1-2 comprises two carbonyl groups each with differentreactivity. This allows one to selectively attach other compounds on thenitrogen atom of the pyrrole ring moiety or the carbonyl carbon of theindoline ring moiety.

Other negatively charged minor groove binders can be readily preparedusing the procedures disclosed herein. For example, as shown in SchemeIII, starting with Compound 1 (or an appropriately protected Compound Aor B), one can readily synthesize a wide variety of chimeric negativelycharged minor groove binders.

Since a wide variety of substituted indoles and other substituted arylcompounds are known, a number of negatively charged minor groove bindersof the present invention can be readily prepared using the methodsdisclosed herein. For example, ethyl5-amino-7-methoxyindole-2-carboxylic acid (Compound A) can be readilyprepared by methods disclosed by Zhang et al. in Synthesis, 1996, 3,377-382. In addition, 3-amino-5-sulfobenzoic acid (Compound B) can besynthesized from 3-nitro-5-sulfobenzoic acid using methods disclosed byVan Dorssen in Recl. Trav. Chim. Pays. Bas., 1910, 29, 376.

In addition, methods for synthesizing neutral or basic compounds (e.g.,Compound C) having structures similar to negatively charged minor groovebinders of the present invention are known to one skilled in the art.See, for example, J. Amer. Chem. Soc., 2000, 122, 6382-6394, which isincorporated by reference in its entirety. Such procedures can be usedto prepare other negatively charged minor groove binders of the presentinvention.

Thus, a wide variety of methods are available for synthesizingnegatively charged minor groove binders of the present invention.

III. Oligonucleotide NMGB Conjugate

Another aspect of the present invention provides anoligonucleotide-negatively charged minor groove binder conjugate, orsimply the “conjugate”. The conjugates of the present invention comprisea negatively charged minor groove binder moiety and an oligonucleotidemoiety which is covalently attached to the negatively charged minorgroove binder moiety optionally through a linker.

Typically, the oligonucleotide moiety comprises from about 3 to about100 nucleotide units. The oligonucleotide can be natural, such as DNAand RNA, unnatural, i.e., synthetic, such as PNA, locked nucleic acid,or a modified DNA and RNA containing modified nucleosides, or acombination thereof. Exemplary locked nucleic acids are disclosed in PCTPublication No. WO 01/56746, which is incorporated herein by referencein its entirety. Exemplary modified DNA and RNA containing modifiednucleosides are disclosed in U.S. patent application Ser. No.09/796,988, which is incorporated herein by reference in its entirety.Syntheses of PNA and PNA/DNA chimeras are known in the art and can beprepared from methods disclosed in, for example, Uhlmann et al., Angew.Chem. Inter. Ed., 1998, 37, 2796-2823 and Mayfield et al., Anal.Biochem., 1998, 401-404, which are incorporated herein by reference intheir entirety.

In a preferred embodiment, the negatively charged minor groove bindermoiety is covalently attached to either the 3′- or 5′-end of theoligonucleotide. Such attachment can be through a terminal base, sugaror phosphate moiety, or through a tail moiety attached to one of thesemoieties. In additional embodiments, the negatively charged minor groovebinder moiety is attached to a nucleotide in an internal position, e.g.,to an internal sugar moiety, or preferably to a base portion of thenucleotide.

In addition, the oligonucleotide-negatively charged minor groove binderconjugates of the present invention can also include other usefulmoieties, such as a fluorophore and/or a quencher. Thus, in oneparticular embodiment of the present invention, theoligonucleotide-negatively charged minor groove binder conjugate is ofthe formula:

where NMGB, ODN, FL, Q, L¹, L², a₁, b₁, c₁ and d₁ are those definedherein.

In one embodiment of the present invention, the NMGB moiety is of theformula:

where n, X³, R³, R⁴, R⁵ and R⁶ are those defined herein.

In another embodiment, the NMGB moiety is of the formula:

where p, R¹⁸, R¹⁹ and R²⁰ are those defined herein, provided at leastone of R¹⁸, R¹⁹ or R²⁰ is the acidic moiety optionally comprising theacidic moiety linker, and one of R¹⁸ and R²⁰ is a point of attachment tothe first linker L¹ or to ODN depending on whether the first linker ispresent or absent, respectively.

In yet another embodiment, the NMGB moiety is of the formula:

where Ar¹, R²¹, R²², R²³ and R²⁴ are those defined herein, provided whenR²³ is hydrogen at least one of Ar¹ or R²¹ is substituted with theacidic moiety optionally comprising the acidic moiety linker, and one ofR²¹, R²², R²³ and R²⁴ is a point of attachment to the first linker L¹ orthe oligonucleotide depending on the presence or absence of the firstlinker, respectively.A. Fluorophores

The fluorophore moieties (FL) are well known to one skilled in the art.See, for example, U.S. Pat. No. 6,114,518, U.S. patent application Ser.No. 09/457,616, and Haugland, et al., HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH CHEMICALS, SIXTH ED., Molecular Probes, Eugene, Oreg. 1996, allof which are incorporated herein by reference in their entirety.Generally, any conventional fluorophore moieties known to one skilled inthe art can be used in the oligonucleotide-negatively charged minorgroove binder conjugates of the present invention. Preferably,fluorophores of the present invention have emission wavelength fromabout 400 to about 1000 nm, more preferably from 400 to about 900 nm,and most preferably from 400 to about 800 nm.

In one particular embodiment, the fluorophore moiety is a latentfluorophore. Such latent fluorophores are used in detection of nucleicacids by hybridization-triggered fluorescence. A latent fluorophore is amolecule in which a physical property of the fluorophore is altered byits interaction with duplex or triplex nucleic acids, resulting in achange in the fluorescence spectrum and/or an increase in thefluorescence quantum yield at a particular wavelength, and/or a changein some other fluorescent property of the molecule. A change influorescence spectrum can include a change in the absorption spectrumand/or a change in the emission spectrum. Such fluorophores aredisclosed in a commonly assigned U.S. patent application Ser. No.09/428,236, filed on Oct. 26, 1999, and entitled“Hybridization-triggered fluorescent detection of nucleic acids,” whichis incorporated herein by reference in its entirety.

In general, the fluorophore moiety can be any polycyclic, preferablypolyaryl, compound which has the emission wave length described herein,for example, fluorescein, rhodamine, bodipy, cyanine, resorufin,coumarin and analogs or derivatives thereof Specific exemplaryfluorophores include analogs or derivatives of fluoresceine, such ascarboxyfluorescein , tetrachlorofluorescein, JOE, HEX, VIC, NED,tetramethylrhodamine, and ROX; cyanine, such as Cy3 and Cy5; resorufin;and coumarin. A fluorophore moiety can be covalently attached to anoligonucleotide through any of a variety of methods known to one skilledin the art. For example, the fluorophore moiety can be converted to afluorophore phosphoramidite reagent of a general formula:FL-L²-X¹⁰where FL and L² are the fluorophore moiety and the second linker asdefined herein, respectively, and X¹⁰ is a reactive group (e.g.,pentafluorophenyl ester or a phosphoramidite). Typically, an activatedfluorophore phosphoramidite reagent of the formula:

is often used to covalently link the fluorophore moiety to the 3′- or5′-end of the oligonucleotide, or to an internal sugar moiety of theoligonucleotide using a conventional synthetic procedure known to oneskilled in the art.

In one particular embodiment of the present invention, fluorophoremoiety is a coumarin derivative selected from the group consisting of:

where R⁶¹, R⁶², R⁶³, R⁶⁴, R⁶⁵, R⁶⁶ and R⁶⁷ are those defined herein,provided that at least one of R⁶¹, R⁶², R⁶³, R⁶⁴, R⁶⁵, R⁶⁶ and R⁶⁷ is abond that attaches the fluorophore to the second linker L² or to theoligonucleotide ODN depending on the presence or absence of the secondlinker, respectively.Synthesis of Fluorophores

In one embodiment, the fluorophore moiety comprises a coumarinchromophore, e.g., moiety of Formula FL-1 above, which are well known toone skilled in the art. Such fluorophore can be covalently attached byusing a corresponding phosphoramidite derivative. See, for example, PCTPublication No. WO 01/42505; Bull. Chem. Soc. Japan. 71 (7):1719-1724(1998); Kartha, et al., Proc. Indian Acad. Sci. Sect. A, 18:28 (1943);Atta, et al., Phosphorus, Sulfur, Silicon Relat. Elem. 80:109-116(1993); U.S. Pat. No. 5,696,157; Nicolaides, et al., J. Chem. Soc.Perkin Trans. I, 2:283-290 (1992); and Saleh, et al., Phosphorus,Sulfur, Silicon Relat. Elem. 48:285-288 (1990). Other useful fluorophoremoieties include 7-hydroxy-3H-phenoxazin-3-one chromophores, which arebased on resorufin core structure that has emission wavelength of 595nm. See, for example, PCT Publication No. WO 01/42505; Forchiassin etal., J. Heterocyc. Chem. 1983, 20, 493-494. Other useful fluorophoresinclude phenoxazine and phenothiaxine derivatives, as well as thoseknown to one skilled in the art.

Additional compounds suitable for elaboration into the presentresorufin-type phosphoramidite reagents, which are suitable forincorporating into the oligonucleotide-negatively charged minor groovebinder conjugates, are described in, for example, co-pending applicationSer. No. 09/457,616; U.S. Pat. No. 4,954,630; Pashkevich, et al., Chem.Heterocycl. Cmpd., Engl. Transl. 11:308-312 (1975); Morrison, et al.,Photochem. Photobiol., 66:245-252 (1997); Afans'eva, et al., Chem.Heterocycl. Cmpd., Engl. Transl. 174-177 (1983); Chem. Abstracts 16329(1955); Long, et al., J. Heterocycl. Chem. 36:895-900 (1999); and Musso,et al., Chem. Ber., 96:1936-1944 (1963), all of which are incorporatedby reference in their entireties.

A number of FL-1 and FL-2 fluorophores are available with analkylcarboxyl group substituent which serves as a starting material forthe synthesis of the corresponding phosphoramidite reagents.Alternatively, these compounds can be converted to reactive esters,e.g., pentafluorophenyl esters. The activated esters can be used tocovalently attach these compounds to amine modified oligonucleotides.

Particular examples of coumarin based fluorophore phosphoramidites thatare useful in conjugates of the present invention include, but are notlimited to, those shown below.

While the oligonucleotide-negatively charged minor groove binderconjugates of the present invention are illustrated in connection withthe fluorophores and quenchers described herein, the present inventionis not limited to these fluorphores and quenchers discussed herein. Anyconventional fluorophores and quenchers known to one skilled in the artis within the scope of the present invention. For example, othersuitable fluorophores and quenchers are disclosed in U.S. Pat. Nos.5,328,824 and 5,824,796, which are incorporated herein by reference intheir entireties.

B. Quenchers

The quencher moieties (Q) are also well known to one skilled in the artand are disclosed, for example, in the above incorporated U.S. Pat. No.6,114,518, U.S. patent application Ser. No. 09/457,616, and Haugland, etal., HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, SIXTH ED.,Molecular Probes, Eugene, Oreg. 1996. Generally, any conventionalquencher moieties known to one skilled in the art can be used in theoligonucleotide-negatively charged minor groove binder conjugates of thepresent invention. Preferably, quencher moieties of the presentinvention have absorbance maximum of from about 400 to about 1000 nm,more preferably from 400 to about 900 nm, and most preferably from 400to about 800 nm. Exemplary quencher moieties include TAMARA, dabcyl anddabsyl having an absorption maximum in the wavelength region of about400 to 800 nm; substituted diphenyldiazenes, such as those disclosed inPCT Publication No. WO 01/42505 and U.S. patent application Ser. No.09/876,830; and substituted phenyl[4-(phenyldiazenyl)-phenyl]diazenes,such as those disclosed in PCT Publication No. WO 01/86001.

In one embodiment, the quencher moiety is a diazo compound of theformula:

where Y, R^(z), R^(w), z and w are those defined herein. When Y issubstituted phenyldiazenyl, the diazo quencher moiety comprises aplurality of diazo groups. For example, one particular diazo quenchermoiety where Y is substituted phenyldiazenyl is of the formula:

wherein Y, z, w, R^(w) and R^(z) are those defined herein.

Substituted diazenyl can be synthesized using methods known to oneskilled in the art. See, for example, PCT Publication No. WO 01/42505and U.S. patent application Ser. No. 09/876,830. Substituted phenyldiazenyl can also be synthesized using methods known to one skilled inthe art. See, for example, PCT Publication No. WO 01/86001. Additionalsubstituted structures with different combinations of substituents atvarious positions can also be prepared, for example, by using compoundsand methods known in the dye chemistry field. See, for example, theColor Index, Issue 3 on CD-ROM, pages 4009-4324; Society of Dyers andColourists, Bradford, England; http://www.sdc.org.uk.

Particular examples of resorufin based quencher phosphoramidites thatare useful in conjugates of the present invention include, but are notlimited to, those shown below.

For those embodiments in which two adjacent R^(w)'s and carbon atoms towhich they are attached form a fused ring system, the linking group L¹of Formula X can be attached to either the phenyl ring (as indicatedabove) or to the ring formed by two adjacent R^(w)'s. Additionally, forthose embodiments herein, where two alkyl groups are attached to anitrogen atom, forming a dialkylamino substituent, the alkyl groups canbe the same or different.

Preferably, the quencher moiety is of the formula:

where w, z, R^(w) and R^(z) are those defined herein; and Y is selectedfrom the group consisting of nitro and —N(CH₃)₂.Synthesis of Quenchers Comprising L¹ Linker

Typically, quenchers of the present invention comprise the linker L¹,infra. In some instances, the linker comprises a phosphoramidite groupand a protected alcohol group which serve as attachment points to anegatively charged minor groove binder and/or an oligonucleotide.Syntheses of these quenchers comprising a linker are illustrated inSchemes IV and V.

As shown in Scheme IV, a substituted 4-(phenyldiazenyl)phenylamine 1,where q is 1 to 20, is reacted with p-nitrophenylchloroformate to yieldCompound 2. Compound 1 in Scheme IV is commercially available or can beproduced using any of the conventional methods known to one skilled inthe art. See, for example, U.S. Pat. No. 2,264,303, which isincorporated herein by reference in its entirety. In Scheme IV, thequencher is shown with linker L¹. In particular, the linker comprises atrifunctional pyrrolidinediol. Example of other L¹ linkers are discussedin detail below and are disclosed in U.S. Pat. No. 5,512,667, which isincorporated herein by reference in its entirety.

Reaction of Compound 2 with a substituted pyrrolidinediol yields a diol3, where R^(x) is a linker having from 1 to 15 chain atoms. The primaryhydroxyl group of diol 3 is protected by reacting with dimethoxytritylchloride (DMTrCl) to yield Compound 4. The secondary hydroxyl group ofCompound 4 is reacted with 2-cyanoethyl diisopropylchlorophosphoramiditeto give the dimethoxytrityl protected phosphoramidite reagent 5. Thedimethoxytrityl protected phosphoramidite reagent 5 can then becovalently linked to an oligonucleotide using methods conventionallyknown to one skilled in the art.

Procedure of Scheme IV is applicable for preparing quenchers with otherL¹ linking groups. For example, by replacing the pyrrolidinediol withacyclic aminodiol compounds, phosphoramidites of Q-1 and Q-2 can beprepared, where r, s, t and v are each independently integers from 1 to20; X is —O— or —CH₂—.

Scheme V illustrates synthesis of another exemplary phosphoramiditequencher 10. Compound 6 is commercially available or can be readilyprepared according to procedures known to one skilled in the art. InScheme V, Compound 6 is reacted with pentafluorophenyl trifluoroacetateto produce an activated ester 7. The activated ester which is thenreacted with a pyrrolidinediol compound having a free primary and a freesecondary hydroxyl group to yield Compound 8. Treatment of Compound 8with DMTrCl followed by reaction with 2-cyanoethyldiisopropylchlorophosphoramidite gives the dimethoxytrityl protectedphosphoramidite reagent 10.

As discussed above, quencher/linker combination of Q-3 can be preparedusing the procedure similar to that of Scheme V by starting with asubstituted 4-(phenyldiazenyl)phenylamine (compound 6) and using anon-cyclic reagent (having an amino and two hydroxyl functions) insteadof the pyrrolidinediol.

Quenchers Attached to a Solid Support Through a Linker

In some aspects of the present invention, the quencher comprising L¹linker is attached to a solid support, for example, controlled poreglass (CPG). This allows solid phase oligonucleotide synthesis. Thelinker has a hydroxyl function that is protected, usually by adimethoxytrityl group which is removed during the synthesis when thefirst nucleotide is attached to the linker. Generally, the samequencher/linker intermediates described above in Schemes IV and V canalso be used to prepare these reagents. One such method is illustratedin Scheme VI.

As shown in Scheme VI, the secondary hydroxyl group of Compound 4 inScheme IV is reacted with succinic anhydride, and thereafter withpentafluorophenyl trifluoroacetate to provide an activated ester 11. Theactivated ester 11 is then reacted with the free amino group attached tothe solid support (e.g., CPG bead) to provide the modified solid support12. The exemplary modified solid support-bound quencher 12 includes a“linker” derived from pyrrolidine diol. However, it should beappreciated that other linkers and related structures, such as thelinkers shown in Q-1, Q-2 and Q-3, can also be used in the procedureillustrated in Scheme VI to provide other solid support-bound quenchers,such as those shown in Q-5 and Q-6.

The solid support-bound quencher 12 in Scheme VI, Q-5 and Q-6 are usefulfor preparing 3′-quencher conjugates, which in turn allow theintroduction of a fluorophore at the 5′-end with the appropriatephosphoramidite or with a fluorophore containing a reactive group. Itshould be appreciated that other solid supports (such as polystyrene)and other cleavable linker systems (in addition to the succinate linkershown) can also be prepared in accordance with these general teachingsand are also within the scope of the invention.

The reaction schemes provided above can be adapted by one of skill inthe art to incorporate a variety of diazo quencher compounds andlinkers. In addition, other structurally related compounds can bereadily modified to produce a variety of quencher compounds using knownchemical reactions. See, for example, Thiel, et al., J. fur prakt.Chemie, 328:497-514 (1986); U.S. Pat. Nos. 4,324,721 and 4,054,560;Timm, Melliand Textilberichte, 9:1090-1096 (1969); Hallas, J.S.D.C.285-294 (1979); Beyer, et al., J Prakt. Chem., 24:100-104 (1964);Hutchings, et al., Chem. Europ. J. 3:1719-1727 (1997) and Morley, etal., J. Phys. Chem. A., 102:5802-5808 (1998); Haak, et al., J. Chem.Res. Miniprint 10:2701-2735 (1998) and Ruggli et al., Helv. Chim. Acta,26:814-826 (1943), all of which are incorporated herein by reference intheir entirety. Furthermore, structures with different combinations ofsubstituents at various positions can also be prepared based oncompounds and methods known in the dye chemistry field. See, forexample, Color Index, Issue 3 on CDD-ROM, pages 4009-4324; Society ofDyers and Colourists, Bradford, England; http://www.sdc.org.uk, all ofwhich are incorporated herein by reference in their entirety.

Some of the quencher which are readily available includedabcylnitrothiazole (e.g., Dabcyl®), QSY® (e.g., QSY-7, MolecularProbes, Eugene Oreg.), Rhodamine, Black Hole Quenchers (BiosearchTechnologies, Novato, Calif.), tetramethylrhodamine,6-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino) hexanoic acid , and6-carboxy-X-rhodamine (Rox).

C. Linkers

Linkers are a chain of atoms that is used to assemble various portionsof the molecule or to covalently attach the molecule (or portionsthereof) to each other or to a solid support. Typically a linker has twoor more, preferably two to four, and more preferably three or four,functional groups that are used to interact with and form covalent bondswith functional groups in the ligands or components, e.g., fluorophores,oligonucleotides, negatively charged minor groove binders, or quenchers.Linkers comprise a chain of atom(s) containing 1 to about 100 atoms,where each chain atom is independently selected from the groupconsisting of C, N, O, S, Si and P. In addition, each chain atom can besubstituted with appropriate substituents known to one skilled in theart. For example, carbon atom can be substituted with a hydrogen,carbonyl oxygen, alkoxy, halide, amine, amide, cyano, hydroxyl. Andsulfur and phosphorous atoms can be substituted with one or more oxygenatoms.

The linker can be acyclic, cyclic, or a combination thereof.Conventional di-, tri-, and tetra-functional linkers are well known toone skilled in the art. For example, exemplary 3′-alkylamine linkers aredescribed in U.S. Pat. No. 5,419,966; exemplary prolinol linkers aredescribed in U.S. Pat. No. 5,512,667; other exemplary tri- andtetrafunctional linkers are described in U.S. Pat. Nos. 5,451,463,5,942,610 and 5,696,251. Some trifunctional linkers are commerciallyavailable, for example, from Glen Research (Sterling, Va.).

Typically, the linking groups are sufficiently robust so that they arestable to reaction conditions used in oligonucleotide synthesis, as wellas the protection/deprotection chemistries used to prepare theconjugates described herein. Suitable linkers are well known to oneskilled in the art. See, for example, U.S. Pat. No. 5,512,667, (prolinolbased linkers), U.S. Pat. Nos. 5,451,463 and 5,141,813, (acycliclinkers), and U.S. Pat. Nos. 5,696,251, 5,585,422 and 6,031,091,(tetrafunctional linking groups), all of which are incorporated hereinby reference in their entireties. Suitable functional groups on linkersfor covalently attaching each component include, but are not limited to,primary and secondary nitrogen, —OH and —SH, and carbonyl derivatives(e.g., esters, anhydrides, amides, acids, carbamates, urea, carbonates,etc.).

Typically, the linker L² can be essentially any linking group thatprovides sufficient spacing for reaction of the phosphoramidite moietyto proceed when the composition is used to introduce FL into anoligonucleotide conjugate or composition. A variety ofheterobifunctional linking groups are commercially available and can beused in the present invention.

It should be appreciated that each of the first linkers, L¹, describedabove represents a common core-structure. As such, the first linkersdescribed above can further comprise additional linkers between thepoint of attachment, as indicated by a squiggly line, and a portion ofthe conjugate to which it is attached. Moreover, the functional groupswhich forms a link can be part of the portion of the conjugate to whichthe linker is attached. In some cases, L¹ is present on the quenchermoiety during its preparation.

In one particular embodiment, the oligonucleotide-negatively chargedminor groove binder conjugate is of the formula:

where

-   -   NMGB, ODN, FL, and Q are those defined herein;    -   each of L_(xx) and L_(yy) is independently a linker comprising a        chain of 2 to 100 chain atoms, wherein each chain atom is        independently selected from the group consisting of C, N, O, P,        and S, provided that the total chain atoms of L_(xx) and L_(yy)        is 100 or less; and    -   k is 0 or 1.

In one particular embodiment, L¹ comprises a moiety selected from thegroup consisting of:

-   -   where a₂ and b₂ are those defined herein;

-   -   where a₂, b₂, c₂, X, R^(g) and R^(h) are those defined herein;        and

-   -   where a₂, b₂, c₂, X, and R^(g) are those defined herein.

Attachment of Q, ODN and NMGB can be any attachment point indicated by asquiggly line. Thus, each linkers above can have a wide variety ofcombinations of Q, ODN and NMGB attachments. In addition, the abovedescribed linker moieties can further comprise additional linkerscovalently attached to the heteroatom attachment point. For example, oneor more components, i.e, Q, ODN and/or NMGB, can be attached to thelinkers shown above at the attachment point indicated, or they can befurther tethered to another linking group.

In one particular embodiment, the first linker, L¹, is preferably amoiety of Formula L-1 or L-2.

In one embodiment, Formula L-1 is a particularly preferred linker whenthe quencher moiety is absent. In another embodiment, Formula L-2 is aparticularly preferred linker when the quencher moiety is present.

In one particular embodiment of the present invention, theoligonucleotide-negatively charged minor groove binder conjugate is ofthe formula:

where

-   -   c₁, d₁, L², Q, ODN, NMGB, and FL are those defined herein;    -   each of a₃, b₃, and c₃ is independently 0 or 1; and

each of L^(a), L^(b) and L^(c) is independently a linker comprising anacyclic chain of from 1 to about 10 atoms, wherein each chain atom isindependently selected from the group consisting of C, N, O, S and P.

In another embodiment of the present invention, theoligonucleotide-negatively charged minor groove binder conjugate is ofthe formula:

where a₂, b₂ c₁, d₁, L², FL, Q, ODN and NMGB are those defined herein.

Still in another embodiment of the present invention, theoligonucleotide-negatively charged minor groove binder conjugate is ofthe formula:

where R^(g), R^(h), X, a₂, b₂, c₂, Q, ODN, NMGB, FL, L², c₁ and d₁ arethose defined herein.

Yet in another embodiment of the present invention, theoligonucleotide-negatively charged minor groove binder conjugate is ofthe formula:

where R^(g), X, a₂, b₂, c₂, Q, ODN, NMGB, FL, L², c₁ and d₁ are thosedefined herein.D. Oligonucleotides

Preferably, an oligonucleotide comprises a plurality of nucleotideunits, a 3′-end and a 5′-end. The oligonucleotide can contain one ormore modified bases other than the normal purine and pyrimidine bases,as well as modified internucleotide linkages capable of specificallybinding target polynucleotide through Watson-Crick base pairing, or thelike. In addition, oligonucleotides can include peptide oligonucleotides(PNAs) or PNA/DNA chimeras, the synthesis of which is known. See, forexample, Uhlmann et al., Angew. Chem. Inter. Ed., 37:2796-2823 (1998);Mayfield et al., Anal. Biochem., 401-404 (1998); and Kyaemo, et al., J.Org. Chem. 65:5167-5176 (2000), all of which are incorporated herein byreference in their entirety.

Preferably, the oligonucleotide has a sufficient number of phophodiesterlinkages adjacent to the 5′ end to allow 5′-3′ exonuclease activity toallow efficient cleavage between the quencher and fluorophore componentsin the conjugate. A suitable number of phosphodiester linkages in thisregard is approximately between 1 and 100, but preferably between 3 and40. In other embodiments, conjugates containing fluorophore and quencherpairs will provide adequate signal upon hybridization to the targetnucleic acid, with cleavage of the probe. Amplified material can bedetected with 5′-MGB-Q-ODN-FL conjugates in which the target isamplified via PCR and the detection is performed in real-time withoutcleavage of the conjugate. This method can also be used as an endpointassay rather than a real-time procedure.

Similarly, modified sugars or sugar analogues can be present in one ormore of the nucleotide subunits of an oligonucleotide conjugate. Sugarmodifications include, but are not limited to, attachment ofsubstituents to the 2′, 3′ and/or 4′ position of the sugar, differentepimeric forms of the sugar, differences in the α- or β-configuration ofthe glycosidic bond, and other anomeric changes. Sugar moieties include,but are not limited to, pentose, deoxypentose, hexose, deoxyhexose,ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose,cyclopentyl, and locked sugars (e.g., sugars containing other cyclic orbridged system resulting in a conformationally locked ring structure).

Oligonucleotide can also include modified bases, in addition to thenaturally-occurring bases adenine, cytosine, guanine, thymine anduracil. Modified bases are considered to be those that differ from thenaturally-occurring bases by addition or deletion of one or morefunctional groups, differences in the heterocyclic ring structure (i.e.,substitution of carbon for a heteroatom, or vice versa), and/orattachment of one or more linker arm structures to the base. Themodified nucleotides which may be included in the ODN conjugates of theinvention include 7-deazapurines and their derivatives andpyrazolopyrimidines (described in PCT WO 90/14353); and in commonlyassigned U.S. patent application Ser. No. 09/054,630.

Exemplary modified bases include the guanine analogues6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (ppG or PPG) and theadenine analogues 4-amino-1H-pyrazolo[3,4-d]pyrimidine (ppA or PPA).Also of use is the xanthine analogue1H-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7H)-dione (ppX). These baseanalogues, when present in an oligonucleotide, strengthen hybridizationand improve mismatch discrimination. All tautomeric forms ofnaturally-occurring bases, modified bases and base analogues may beincluded in the oligonucleotide conjugates of the invention. Inaddition, modified internucleotide linkages capable of specificallybinding target polynucleotide through Watson-Crick base pairing, or thelike may also be included in the oligonucleotide conjugates of thepresent invention. Oligonucleotides can also include peptideoligonucleotides (PNAs) or PNA/DNA chimeras, the synthesis of which isknown and can be performed for example in accordance with thepublications Uhlmann et al., Angew. Chem. Inter. Ed., 37:2796-2823(1998) and Mayfield et al., Anal. Biochem., 401-404 (1998).

As stated above, modified internucleotide linkages can also be presentin oligonucleotide conjugates of the invention. Such modified linkagesinclude, but are not limited to, peptide, phosphate, phosphodiester,phosphotriester, alkylphosphate, alkanephosphonate, thiophosphate,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, substituted phosphoramidate and the like. Severalfurther modifications of bases, sugars and/or internucleotide linkages,that are compatible with their use in oligonucleotides serving as probesand/or primers, will be apparent to those of skill in the art.

IV. Utility

Minor groove binders are useful in a variety of pharmaceuticalapplications. See, for example, Reddy et al., Pharmacol. & Therap.,1999, 84, 1-111. Briefly, it is well known that certain bis-distamycinsand related lexitropsins show activities against human immunodeficiencyvirus (HIV)-1 and HIV-2 at low nanomolar concentrations. It is also wellrecognized that some furan-containing analogues of berenil play animportant role in their activities against Pneumocystis carinii andCryptosporidium parvuam infections in vivo. Furthermore, Pt-pentamidineshows higher antiproliferative activity against small cell lung,non-small cell lung, and melanoma cancer cell lines compared with manyother tumor cell lines. Moreover, trans-butenamidine shows good anti-P.carinii activity in rats. Pentamidine is used against P. cariniipneumonia in individuals infected with HIV who are at high risk fromthis infection. Neothramycin is used clinically for the treatment ofsuperficial carcinoma of the bladder. Turner and Denny, Current DrugTargets, 2000, 1, 1-14 reported on the potential of sequence specificminor groove binders in the treatment of human disease. These compoundsact in a variety of ways to inhibit gene expression, DNA replication andalso alter nuclear architecture. Hybrid molecules containingpyrrolo[2,1-c]benzodiazepine and minor-groove-binding oligopyrrolecarriers showed in vitro antiproliverative activity of K562 and Jurkatcell lines. See Baraldi et al., J. Med. Chem., 1999, 42, 5131-5141.Positively charged netropsin derivatives has been described in WO01/74898, these compounds have multiple applications, including use inhuman and animal medicine and in agriculture. Please note that chargedminor groove binders show biological activity (Reddy et al)

The compositions (i.e., conjugates) of the present invention can be usedwith a variety of techniques, both currently in use and to be developed,in which hybridization of an oligonucleotide to another nucleic acid isinvolved. These include, but are not limited to, techniques in whichhybridization of an oligonucleotide to a target nucleic acid is theendpoint; techniques in which hybridization of one or moreoligonucleotides to a target nucleic acid precedes one or morepolymerase-mediated elongation steps which use the oligonucleotide as aprimer and the target nucleic acid as a template; techniques in whichhybridization of an oligonucleotide to a target nucleic acid is used toblock extension of another primer; techniques in which hybridization ofan oligonucleotide to a target nucleic acid is followed by hydrolysis ofthe oligonucleotide to release an attached label; and techniques inwhich two or more oligonucleotides are hybridized to a target nucleicacid and interactions between the multiple oligonucleotides aremeasured. The conditions for hybridization of oligonucleotides, and thefactors which influence the degree and specificity of hybridization,such as temperature, ionic strength and solvent composition, arewell-known to those of skill in the art. See, for example, Sambrook etal., supra; Ausubel et al., supra; Innis et al. (eds.) PCR Protocols,Academic Press, San Diego, 1990; Hames et al. (eds.) NUCLEIC ACIDHYBRIDISATION: A PRACTICAL APPROACH, IRL Press, Oxford, 1985; and vanNess et al. (1991) Nucleic Acids Res. 19:5143-5151.

Additionally, the compounds described herein can be used to detectpolymeric targets such a nucleic acids using techniques utilized for,e.g., gene expression, SNP detection, sequencing methods, FRET detection(TaqMan assays, molecular beacons, linear beacons), array-based methods,primer extension, enzymatic methods, and the like.

In one embodiment, one or more FL-oligonucleotide conjugates are used asprobe(s) to identify a target nucleic acid by assaying hybridizationbetween the probe(s) and the target nucleic acid. A probe can be labeledwith any detectable label of the present invention, or it can have thecapacity to become labeled either before or after hybridization, such asby containing a reactive group capable of association with a label or bybeing capable of hybridizing to a secondary labeled probe, either beforeor after hybridization to the target. As a basis of this technique it isnoted that conditions for hybridization of nucleic acid probes arewell-known to those of skill in the art. See, for example, Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, SECOND EDITION, Cold SpringHarbor Laboratory Press (1989); Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons (1987, 1988, 1989, 1990, 1991,1992, 1993, 1994, 1995, 1996); Hames et al. (eds.) NUCLEIC ACIDHYBRIDIZATION: A PRACTICAL APPROACH, IRL Press, Oxford, 1985; and vanNess et al. Nucleic Acids Res. 19:5143-5151(1991).

Hybridization can be assayed (i.e., hybridized nucleic acids can beidentified) by distinguishing hybridized probe from free probe by one ofseveral methods that are well-known to those of skill in the art. Theseinclude, but are not limited to, attachment of target nucleic acid to asolid support, either directly or indirectly (by hybridization to asecond, support-bound probe or interaction between surface-bound andprobe-conjugated ligands) followed by direct or indirect hybridizationwith probe, and washing to remove unhybridized probe; determination ofnuclease resistance; buoyant density determination; affinity methodsspecific for nucleic acid duplexes (e.g., hydroxyapatitechromatography); interactions between multiple probes hybridized to thesame target nucleic acid; and other known techniques. See, for example,Falkow et al., U.S. Pat. No. 4,358,535; Urdea et al., U.S. Pat. Nos.4,868,105 and 5,124,246; Freifelder, PHYSICAL BIOCHEMISTRY, SECONDEDITION, Freeman & Co., San Francisco, 1982; Sambrook, et al., supra;Ausubel et al., supra; and Hames et al., supra.

Other applications for oligonucleotide-negatively charged minor groovebinder conjugates comprising a fluorophore and quencher are found inassays in which a labeled probe is hybridized to a target and/or anextension product of a target, and a change in the physical state of thelabel is effected as a consequence of hybridization. A probe is anucleic acid molecule that is capable of hybridizing to a targetsequence in a second nucleic acid molecule. By way of example, one assayof this type, the hydrolyzable probe assay, takes advantage of the factthat many polymerizing enzymes, such as DNA polymerases, possessintrinsic 5′-3′ exonucleolytic activities. Accordingly, if a probe ishybridized to a sequence that can serve as a template for polymerization(for instance, if a probe is hybridized to a region of DNA locatedbetween two amplification primers, during the course of an amplificationreaction), a polymerizing enzyme that has initiated polymerization at anupstream amplification primer is capable of exonucleolytically digestingthe probe. Any label attached to such a probe will be released, if theprobe is hybridized to its target and if amplification is occurringacross the region to which the probe is hybridized. Released label isseparated from labeled probe and detected by methods well-known to thoseof skill in the art, depending on the nature of the label. For example,radioactively labeled fragments can be separated by thin-layerchromatography and detected by autoradiography; whilefluorescently-labeled fragments can be detected by irradiation at theappropriate excitation wavelengths with observation at the appropriateemission wavelengths. This basic technique is described for example inU.S. Pat. No. 5,210,015.

A probe can contains both a fluorescent label and a quenching agent,which quenches the fluorescence emission of the fluorescent label. Inthis case, the fluorescent label is not detectable until its spatialrelationship to the quenching agent has been altered, for example byexonucleolytic release of the fluorescent label from the probe. Thus,prior to hybridization to its target sequence, the dualfluorophore/quencher labeled probe does not emit fluorescence.Subsequent to hybridization of the fluorophore/quencher-labeled probe toits target, it becomes a substrate for the exonucleolytic activity of apolymerizing enzyme which has initiated polymerization at an upstreamprimer. Exonucleolytic degradation of the probe releases the fluorescentlabel from the probe, and hence from the vicinity of the quenchingagent, allowing detection of a fluorescent signal upon irradiation atthe appropriate excitation wavelengths. This method has the advantagethat released label does not have to be separated from intact probe.Multiplex approaches utilize multiple probes, each of which iscomplementary to a different target sequence and carries adistinguishable label, allowing the assay of several target sequencessimultaneously.

Another application embodiment uses a self-probing primer with anintegral tail, where the quencher/fluorophore is present in the hairpin,that can probe the extension product of the primer and afteramplification hybridizes to the amplicon in a form that fluoresces. Theprobing of a target sequence can thereby be converted into aunimolecular event (Whitcombe, et al., Nat. Biotech., 17:804-807(1999)).

Compositions of the invention can also be used in various techniqueswhich involve multiple fluorescent-labeled probes. In some of theseassays, changes in properties of a fluorescent label are used to monitorhybridization. For example, fluorescence resonance energy transfer(FRET) has been used as an indicator of oligonucleotide hybridization.In one embodiment of this technique, two probes are used, eachcontaining a fluorescent label and a quencher molecule respectively. Thefluorescent label is a donor, and the quencher is an acceptor, whereinthe emission wavelengths of the donor overlap the absorption wavelengthsof the acceptor. The sequences of the probes are selected so that theyhybridize to adjacent regions of a target nucleic acid, thereby bringingthe fluorescence donor and the acceptor into close proximity, if targetis present. In the presence of target nucleic acid, irradiation atwavelengths corresponding to the absorption wavelengths of thefluorescence donor will result in emission from the fluorescenceacceptor. These types of assays have the advantage that they arehomogeneous assays, providing a positive signal without the necessity ofremoving unreacted probe. For further details and additional examples ofthe assays which are known in the art, see, for example, European PatentPublication 070685; Agrawal & Zamecnik, Nucl. Acids Res., 18:5419-5423(1990); and Cardullo et al. (1988) Proc. Natl. Acad. Sci. USA 85:8790-8794. Additional applications of the novel compositions of thepresent invention are in those and related techniques in whichinteractions between two different oligonucleotides that are bothhybridized to the same target nucleic acid are measured. The selectionof appropriate fluorescence donor/fluorescence acceptor pairs will beapparent to one of skill in the art, based on the principle that, for agiven pair, the emission wavelengths of the fluorescence donor willoverlap the absorption wavelengths of the acceptor.

In another application of the novel compositions of the invention, thefluorescence of the conjugate is quenched in its native state. But,after hybridization with the intended target the spatial arrangement ofthe fluorophore and quencher moieties are changed such that fluorescenceoccurs. For this basic technique see, for example, Tyagi et al., Nat.Biotech., 16:49-53 (1998); and U.S. Pat. No. 5,876,930.

It should be understood that in addition to the fluorophores which arefound in accordance with the present invention especially useful to beused with the quenchers of the invention, and which fluorophores areincorporated into ODNs in accordance with the invention, a person ofordinary skill can choose additional fluorophores to be used incombination with the quenchers of the present invention, based on theoptical properties described in the literature, such as the references:Haugland HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, SixEdition, Eugene, Oreg. pp. 235-236. 1996; Berlman, Handbook ofFluorescence Spectra of Aromatic Molecules, 2^(nd), Academic Press, NewYork, 1971; Du et al., PhotochemCAD. A Computer-Aided Design andResearch Tool in Photochemistry, Photochem. Photobiol. 68:141-142(1998).

In another application, the minor groove binder,dihydrocyclopyrroloindole tripeptide (i.e., DPI₃ or CDPI₃), is coupledto a quencher in a FL-ODN-Q-CDPI₃ conjugate to improve signal to noiseratios.

Yet another application of the conjugates of the present invention is toincorporate the pair into enzyme substrates, where fluorescence isquenched because of the proximity of the fluorophore and quencher.However, after an enzyme cleaves the substrate the fluorophore andquencher become separated and fluorescence is observed. It should beappreciated that conjugates containing both the quenchers andfluorophores can be constructed such that they are cleavedenzymatically.

Still in another application, the oligonucleotide conjugates of thepresent invention are utilized in procedures employing arrays ofoligonucleotides or oligonucleotide conjugates. Techniques forsequencing by hybridization, single nucleotide polymorphism analysis(SNPs) and array-based analysis of gene expression (see, Hacia, et al.,Nat. Genet. 22:119-120 (1999)) are well-known and can be readily adaptedto utilize the conjugates of the present invention. For example, anordered array of oligonucleotides of different known sequences (or theirconjugates) is used as a platform for hybridization to one or more testpolynucleotides, nucleic acids or nucleic acid populations.Determination of the oligonucleotides which are hybridized and alignmentof their known sequences allows reconstruction of the sequence of thetest polynucleotide. See, for example, U.S. Pat. Nos. 5,492,806;5,525,464; 5,556,752; and PCT Publications WO 92/10588 and WO 96/17957.Materials for construction of arrays include, but are not limited to,nitrocellulose, glass, silicon wafers and optical fibers.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES Example 1

This example illustrates a method for producing3-[(tert-butyl)oxycarbonyl]-7-[(2,3,4,5,6-pentafluoropheny)oxycarbonyl]pyrrolo[3,2-e]indoline-5-sulfonicacid (Compound 1-2 in Scheme II)

Synthesis of 1-acetylindoline-6-Sulfonic Acid (Compound 2-2 in SchemeII)

Indoline-6-sulfonic acid (Compound 2-1 in Scheme II) (20 g), which wasprepared according to the procedure described in U.S. Pat. No.4,405,788, was dissolved in 200 mL of anhydrous DMF in the presence of20 mL of triethylamine. Acetic anhydride (12 mL) was added, and theresulting solution was kept at room temperature for 8 h. DMF wasevaporated and the residue was diluted with 100 mL of water.Concentrated sulfuric acid (20 mL) was added slowly with cooling. Theproduct was allowed to crystallize overnight at 4° C. The crystals werecollected by filtration and washed with small amount of cold waterfollowed by acetone. Drying under vacuum over P₂O₅ afforded 18.6 g ofthe desired product as white crystals. ¹H MNR (d6-DMSO) δ: 8.32 (s, 1H),7.25 (dd, J₁=7.7 Hz, J₂=1.5 Hz, 1H) 7.14 (d, J=7.7 Hz), 6.70 (OH+H₂O),4.07 (t, J=8.5 Hz, 2H), 3.09 (t, J=8.5 Hz, 2H) and 2.14 (s, 3H).

Synthesis of 1-acetyl-5-nitroindoline-6-Sulfonic Acid (Compound 2-3 inScheme II)

16.0 g (66 mmol) of 1-acetylindoline-6-sulfonic acid (Compound 2-2 inScheme II) was dissolved in 160 mL of cone, sulfuric acid. The solutionwas cooled to 0° C. using ice/water bath. Nitric acid (90%, fuming) (3.0mL, 1.1 eqv.) was added dropwise over 10 min. The resulting pale yellowsolution was stirred at 0° C. for 2 h and then slowly poured onto 200 gof ice. The crystals formed were collected by filtration and washedsuccessively with cold water acetone and ether. Drying under vacuum overP₂O₅ afforded 18.2 g of the title compound. ¹H MNR (d6-DMSO) δ: 8.47 (s,1H), 7.40 (s, 1H), 5.54 (OH+H₂O), 4.12 (t, J=8.5 Hz, 2H), 3.13 (t, J=8.5HZ, 2H) and 2.17 (s, 3H). ¹³C NMR (d6-DMSO) δ: 169.19, 144.18, 143.17,139.54, 133.54, 119.10, 115.23, 48,67, 26.75 and 24.01.

Synthesis of 1-aceyl-5-aminoindoline-6-Sulfonic Acid (Compound 2-4 inScheme II)

A suspension of 1-acetyl-5-nitroindoline-6-sulfonic acid (Compound 2-3in Scheme II) (9.0 g) in 250 mL of methanol was hydrogenated in thepresence of 0.5 g of 10% Pd/C for 3 h. DMF (˜250 mL) was added todissolve the precipitated product. The catalyst was removed byfiltration through Celite® and the filtrate was concentrated to give asolid. The solid was suspended in methanol (50 mL), filtered and washedwith small amount of methanol and ether. Drying under vacuum afforded6.4 g of the desired amine as an off-white solid.

5-{[(1E)-1-aza-2-(ethoxycaxbonyl)prop-1-enyl]amino}-1-acetylindoline-6-SulfonicAcid (Compound 2-6 in Scheme II)

A suspension of 1-aceyl-5-aminoindoline-6-sulfonic acid (Compound 2-4 inScheme II) (6.3 g, mmol) in a mixture of conc. HCl (40 mL) and water (35mL) was cooled in ice/water bath. To the stirred mixture was slowly (˜10min) added a solution of NaNO₂ (2.0 g) in 10 mL of water. The resultantyellow suspension of the diazonium salt 5 was stirred at 0° C. foranother 1 h and then used in the next reaction without workup.

To the above suspension of the diazonium salt (Compound 2-5 in SchemeII) was added a solution of SnCl₂ dihydrate (30.0 g) in 50 mL of water.The initially clear solution turned into a thick suspension in about 1min. It was stirred for 30 min and treated with 4 mL of ethyl pyruvateto give a yellow suspension of the crude product (Compound 2-6 in SchemeII). After being stirred for another 30 min the solid was filtered off,washed with a mixture of 20 mL of conc. HCl and 80 mL of water followedby cold water. Drying under vacuum overnight afforded 8.7 g of the crudeproduct (Compound 2-6 in Scheme II) which was contaminated with the tinsalts. This product was 95% pure by HPLC analysis and was used in thenext step without further purification.

Synthesis of 3-acetyl-7-(ethoxycarbonyl)[3,2-e]indoline-5-Sulfonic Acid(Compound 2-7 in Scheme II)

A solution of the crude5-{[(1E)-1-aza-2-(ethoxycaxbonyl)prop-1-enyl]amino}-1-acetylindoline-6-sulfonicacid (Compound 2-6 in Scheme II) (8.5 g) in 220 mL of TFA was refluxedfor 90 min. The reaction mixture was cooled and concentrated. Theresulting black solid was chromatographed on silica eluting with aneluent comprising 5% triethylamine, 10% methanol and 85%dichloromethane. The product containing fractions were combined,concentrated, and the residue was triturated with cold methanol (30 mL).The precipitated off-white solid was collected by filtration, washedwith ethyl acetate/hexane (1:1) and dried under vacuum. The yield of thepure product (triethylammonium salt) was 3.7 g. ¹H MNR (d6-DMSO) δ:10.09 (s, 1H), 8.85 (br s, 1H), 8.46 (s, 1H), 7.12 (d, J=2.2 Hz, 1H),4.36 (q, J=7 Hz, 2H), 4.18 (t, J=8.7 Hz, 2H), 3.31 (t, J=8.6 Hz, 2H),3.09 (m, 6H), 2.16 (s, 3H), 1.34 (t, J=7 Hz, 3H) and 1.16 (t, J=7.4 Hz).

Synthesis of3-[(tert-butyl)oxycarbonyl]-5-sulfopyrrolo[3,2-e]indoline-7-CarboxylicAcid (Compound 2-9 in Scheme II)

To a solution of 3-acetyl-7-(ethoxycarbonyl)[3,2-e]indoline-5-sulfonicacid (Compound 2-7 in Scheme II) (3.6 g) in 37 mL of water was added 23mL of 3 M KOH solution. The resultant suspension was stirred for 15 minto give a clear solution. The flask was placed into an oil bath andheated with stirring at 80° C. for 7 h. The resultant solution of theaminoacid (Compound 2-8 in Scheme II) was cooled to room temperature,and sodium bicarbonate (4.5 g) was added followed by a solution of Bocanhydride (2.0 g) in 20 mL of THF. The resulting emulsion was vigorouslystirred for 5 h and then acidified with citric acid (˜12 g) to pH ofabout 3. The mixture was concentrated to a semi-solid and trituratedwith methanol. The insoluble potassium citrate was filtered off, and thefiltrate was concentrated. The solid was dissolved in DMF (100 mL).After stirring for 1 h, the solution was filtered to remove small amountof residual salts and concentrated. Drying under vacuum afforded 3.5 gof the desired product (Compound 2-9 in Scheme II) as a pale yellow,amorphous solid. The NMR analysis showed that the product wascontaminated with potassium acetate and DMF. ¹H MNR (d6-DMSO) δ: 9.98(s, 1H), 8.05 (br s, 1H), 7.95 (s, 1H, DMF), 7.00 (d, J=2.2 Hz, 1H),4.00 (m, 2H), 3.23 (t, J=8.7 Hz, 2H), 2,80 (d, DMF), 1.96 (s, CH₃COO⁻and 1.51 (s, 911).

Synthesis of3-[(tert-butyl)oxycarbonyl]-7-[(2,3,4,5,6-pentafluoropheny)oxycarbonyl]-pyrrolo[3,2-e]indoline-5-sulfonicacid (Compound 1-2 in Scheme II)

To a solution of the above compound (Compound 2-9 in Scheme II) (1.7 g)in DMF (10 mL) was added triethylamine (2.0 mL) followed bypentafluorophenyl trifluoroacetate (i.e., PFP-TFA) (2.0 mL). Afterstirring for 1 h, the solution was concentrated under vacuum to afford adark oil. The mixture was chromatographed on silica eluting with agradient of methanol (10-20%) in dichloromethane. The product fractionswere combined and concentrated to give a tan, oily product. Triturationwith 30% ethyl acetate/hexane produced a precipitate of the desiredpentafluorophenyl (i.e., PFP) ester (1.22 g). ¹H MNR (d6-DMSO) δ: 10.34(s, 1H), 8.25 (br s, 1H), 8.46 (s, 1H), 7.56 (d, J=2.2 Hz, 1H), 4.04 (tJ=8.7 Hz, 2H), 3.29 (t, J=8.6 Hz, 2H) and 1.53 (s, 911).

Example 2

This example illustrates a method for synthesizing CDPI-trimer (i.e.,CDPI₃) derivative of the present invention.

Synthesis of3-[(tert-butyl)oxycarbonyl]-7-[(7-{[2-4-nitrophenyl)ethyl]oxycarbonyl}-pyrrolo[3,2-e]indolin-3-yl)carbonyl]pyrrolo[3,2-indoline-5-SulfonicAcid, triethylammonium Salt (13)

2-(4-Nitrophenyl)ethyl3-[(tert-butyl)oxycarbonyl]pyrrolo[4,5-e]indoline-7-carboxylate (1.62 g)(Compound 1-1 of Scheme I) was selectively deprotected by treatment with15 mL of trifluoroacetic acid (i.e., TFA) for 1 h. TFA was evaporated toafford a TFA salt of amine 12. Residual TFA was removed byco-evaporation with ether followed by drying under vacuum. The TEA saltwas dissolved in 30 mL of anhydrous DMF in the presence of 0.9 mL oftriethylamine. Compound 1-2 of Scheme II (see Example 1) (1.86 g) wasadded and the reaction was stirred at room temperature overnight. Thesolvent was evaporated under vacuum and the semi-solid residue wastriturated with 10% MeOH/dichloromethane. The resulting yellow solid wascollected by filtration and washed with the MeOH/dichloromethane mixturefollowed by ether. Drying under vacuum afforded 2.2 g of the dimer 13.The product contained about 1 equivalent of DMF according to the NMRanalysis. ¹H MNR (d6-DMSO) δ: 11.96 (s, 1H, NH), 10.27 (s, 1H, NH), 8.84(br s, 1H), 8.33 (d, J=8.8 Hz, 1H), 8.19 (d, J=8.8 Hz, 2H), 8.12 (br s,1H), 7.65 (d, J=8.8 Hz, 2H), 7.35 (d, J=8.8 Hz, 1H), 7.08 (s, 1H), 7.07(s, 1H), 4.64 (t, J=8 Hz, 2H), 4.57 (t, J=6.4 Hz, 2H), 4.03 (t, J=8.5Hz, 2H), 3.44 (t, J=8 Hz, 2H), 3.27 (t, J=8.5 Hz, 2H), 3.22 (t, J=6.6Hz, 2H), 3.09 (m, 6H), 1.52 (s, 9H) and 1.16 (t, J=7.4 Hz, 9H).

Synthesis of3-[(3-carbarmoylpyrrolo[4,5-e]indolin-7-yl)carbonyl]-7-[(7-{[2-(4-nitrophenyl)ethyl]oxycarbonyl}pyrrolo[3,2-e]indolin-3-yl)carbonyl]pyrrolo[3,2-e]indoline-5-SulfonicAcid (Compound 1-4, n=1, X═SO₃H, of Scheme I)

Compound 13 (1.2 g) from above was deprotected by a treatment with 26 mLof 50% TFA/CH₂Cl₂ for 2 h. The reaction was concentrated and theresultant TFA salt was washed with ether and dried under vacuum. To asuspension of the TFA salt in anhydrous DMF (25 mL) was addedtriethylamine (0.9 mL) followed by 2,3,5,6-tetrafluorophenyl3-carbamoylpyrrolo[4,5-e]indoline-7-carboxylate (0.8 g) (prepared fromthe procedure described by Lukhtanov et al. in Bioconjugate Chem., 1995,6, 418-426). The reaction mixture was stirred at room temperature for 24h. DMF was evaporated and the residue was triturated with CH₃CN. Thesolid was stirred overnight and collected by filtration. Washing withethyl acetate and drying under vacuum afforded 1.3 g of the trimer(Compound 1-4, n=1, X═SO₃H, of Scheme I) as an off-white solid.

Synthesis of3-[(3-carbamoylpyrrolo[4,5-e]indolin-7-yl)carbonyl]-7-({7-[(2,3,4,5,6-pentafluorophenyl)oxycarbonyl]pyrrolo[3,2-e]indolin-3-yl}carbonyl)pyrrolo [(3,2-e]indoline-5-Sulfonic Acid (Compound 1-5, n=1,X═SO₃H, of Scheme I)

To a suspension of Compound 1-4 (n=1, X═SO₃H, of Scheme I) (1.3 g) in 75mL of DMF was added 4.5 mL of DBU. After being stirred at 50° C. for 40min the solution was concentrated and the residue was triturated withether (150 mL). The solid was filtered off, washed with ether and dried.This procedure afforded 1.7 g of the free acid (DBU salt) as a paleyellow solid. This product was suspended in anhydrous DMF (35 mL) andtreated with triethylamine (1.7 mL) followed by PEP-TFA (1.7 mL). Afterbeing stirred for 1 h the solution was concentrated, ether was added tothe residue to precipitate the product. It was filtered off, washed with50% MeOH/ether and ether. Drying under vacuum afforded 1.1 g of thetitle PFP ester as a dark yellow solid.

Example 3

This example illustrates a method for synthesizing CDPI-tetramer (i.e.,DPI₄ or CDPI₄) derivative of the present invention.

Synthesis of3-({3-[(tert-butyl)oxycarbonyl]-5-sulfopyrrolo[4,5-e]indolin-7-yl}carbonyl)-7-[(7-{[2-(4-nitrophenyl)ethyl]carbonyl]pyrrolo[3,2-e]indoline-5-sulfonic,ditriethylammonium Salt (16)

A suspension of Compound 13 (2.1 g) (see Example 2) in 20 mL of TFA wasstirred at room temperature for 1 h. TFA was evaporated and theresulting solid was washed with ether to remove residual TFA. Afterbeing dried in vacuo for 1 h, the solid was dissolved in 20 mL ofanhydrous DMF in the presence of 1.0 mL of triethylamine. To thissolution was added Compound 1-2 (Scheme II) (1.6 g) (see Example 1). Theresulting reaction mixture was stirred overnight to give a suspension.DMF was evaporated and the solid was washed with methanol, ether anddried under vacuum to afford 2.8 g of the title Compound 16. ¹H MNR(d6-DMSO) δ: 11.97 (s, 1H, NH), 10.39 (s, IH, NH), 10.29 (s, 1H, NH),8.85 (br s, 2H), 8.58 (s, 1H), 8.34 (d, J=8.7 Hz, 1H), 8.20 (d, J=8.8Hz, 2H), 8.12 (br s, 1H), 7.67 (d, J=8.7 Hz, 2H), 7.36 (d, J=8.8 Hz,1H), 7.22 (d, J=1 Hz, 1H), 7.12 (d, J=1 Hz, 1H), 7.09 (d, J=1 Hz, 1H),4.70 (t, J=8 Hz, 4H), 4.58 (t, J=6.4 Hz, 2H), 4.05 (t, J=8.6 Hz, 2H),3.50 (m, 4H), 3.28 (t, J=8.5 Hz, 2H), 3.23 (t, J=6.6 Hz, 2H), 3.09 (m,12H), 1.53 (s, 9H) and 1.17 (t, J=7.4 Hz, 18H).

Synthesis of3-({3-[(3-carbamoylpyrrolo[4,5-e]indolin-7-yl]-5-sulfopyrrolo[4,5-e]indolin-7-yl}carbonyl)-7-[(7-{[2-(4-nitrophenyl)ethyl]oxycarbonyl}pyrrolo[3,2-indolin-3-yl)carbonyl]pyrrolo[3,2-elindoline-5-SulfuricAcid, ditriethylammonium Salt (Compound 1-4, n=2, X═SO₃H, of Scheme I))

Compound 16 (1.5 g) from above was selectively deprotected by atreatment with 26 mL of 50% TFA/CH₂Cl₂ for 2 h. The reaction wasconcentrated and the resultant TFA salt was washed with ether and driedunder vacuum. To a suspension of the TFA salt in DMF (25 mL) was addedtriethylamine (0.9 mL) followed by 2,3,5,6-tetrafluorophenyl3-carbamoylpyrrolo[4,5-e]indoline-7-carboxylate (0.8 g) (prepared fromthe procedure described by Lukhtanov et al. in Bioconjugate Chem., 1995,6, 418-426). The reaction mixture was stirred at room temperature for 48h. DMF was evaporated and the residue was triturated with CH₃CN. Thesolid was stirred overnight and collected by filtration. Washing withethyl acetate and drying under vacuum afforded 1.6 g of the tetramer(Compound 1-4, n=2, X═SO₃H, of Scheme I) as an off-white solid. ¹H MNR(d6-DMSO) δ: 11.97 (s, 1H, NH), 11.55 (s, 1H, NH), 10.40 (s, 2H, 2NH),8.85 (br s, 2H), 8.60 (s, 1H), 8.57 (s, 1H), 8.35 (d, J=8.9 Hz, 1H),8.20 (d, J=8.7 Hz, 2H), 7.98 (d, J=9 Hz, 1H), 7.67 (d, J=8.7 Hz, 2H),7.36 (d, J=9 Hz, 1H), 7.25 (d, J=9 Hz, 1H), 7.22 (br s, 2H), 7.09 (d,J=1 Hz, 1H), 6.99 (d, J=1 Hz, 1H), 6.11 (br s, 2H, NH₂), 4.70 (m, 6H),4.58 (t, J=6 Hz, 2H), 3.99 (t, J=8.7 Hz, 2H), 3.49 (m, 6H), 3.30 (t,3=8.5 Hz, 2H), 3.23 (t, J=6 Hz, 2H); 3.09 (m, 12H), 1.16 (t, J=7.4 Hz,18H).

Synthesis of3-({3-[(3-carbamoylpyrrolo[4,5-e]indolin-7-yl]-5-sulfopyrrolo[4,5-e]indolin-7-yl}carbonyl)-7-({7-[(2,3,4,5,6-pentafluorophenyl)oxycarbonyl]pyrrolo[3,2-e]indolin]-3-yl}carbonyl)pyrrolo[3,2-e]indoline-5-SulfonicAcid, ditriethylammonium Salt (Compound 1-5, n=2, X═SO₃H, of Scheme I)

To a suspension of Compound 1-4 (n=2, X═SO₃H, of Scheme I) (1.5 g) in 75mL of DMF was added 4.5 mL of DBU. After stirring at 50° C. for 40 min,the solution was concentrated and the residue was triturated with ether(150 mL). The solid was filtered off, washed with ether and dried. Thisprocedure afforded 1.7 g of the free acid (di-DBU salt) as a pale yellowsolid. This product was suspended in anhydrous DMF (35 mL) and treatedwith triethylamine (1.7 mL) followed by PEP-TFA (1.7 mL). After stirringthe resulting mixture for 1 h, the solution was concentrated. To theresidue was added ether to precipitate the product (Compound 1-5, n=2,X═SO₃H, of Scheme I). It was filtered off, washed with 50% MeOH/ether,ether and dried. The yield of the final product was 1.48 g. ¹H MNR(d6-DMSO) δ: 12.54 (s, 1H, NH), 11.54 (s, 1H, NH), 10.41 (s, 2H, 2NH),8.84 (br s, 2H), 8.61 (s, 1H), 8.57 (s, 1H), 8.46 (d, J=8.9 Hz, 1H),7.98 (d, J=9 Hz, 1H), 7.60 (d, J=1 Hz, 1H), 7.44 (d, J=9 Hz, 1H), 7.25(d, J=9 Hz, 1H), 7.23 (br s, 2H), 6.99 (d, J=1 Hz, 1H), 6.10 (br s, 2H,NH₂), 4.70 (m, 6H), 3.99 (t, J=8.5 Hz, 2H), 3.49 (m, 6H), 3.30 (m, 2H),3.09 (m, 12H) and 1.16 (t, J=7.4 Hz, 18H).

Example 4

This example illustrates conjugates of the present invention to performmismatch discrimination.

Oligonucleotide conjugates 5′-CCAAAATTAC-X¹¹-3′ (SEQ ID NO:1), where X¹¹ is a minor groove binder of FIG. 1, were hybridized to differentcomplementary targets that contain A/C mismatch at positions 5, 6, 7 and8, relative to the attached minor groove binder.

The relative free energy difference (ΔΔG°) between match and mismatch atpositions 5, 6, 7 and 8 were calculated at 50° C. and are listed inTable below. The ΔΔG° values were calculated as described in U.S.application Ser. No. 09/796,988.

Table of ΔΔG° between match and mismatch calculated at 50° C. fordifferent conjugates.

A/C Mismatch CDPI₃ (N) CDPI₃ (C) CDPI₃—SO₃ ⁻ Position cal/mol cal/molcal/mol 5 5280 4870 4260 6 6320 6220 5160 7 5550 6240 5550 8 5510 55204320

As shown in the above Table, the CDPI₃-SO₃ ⁻ conjugates performcomparable to the other conjugates in the Table.

In addition, the CDPI₄ moiety when attached to an oligonucleotide yieldsconjugates with aggregation properties that make it not practical touse. However, when the CDPI₄ is substituted with one or more SO₃Hgroups, their aggregation problems are significantly reduced such thatthey can be successfully used in hybridization base studies and assays.

Example 5

This example shows measured thermodynamic properties of conjugates ofthe present invention.

Thermodynamic properties of 3′ -conjugated, where X″ represents theattached minor groove binder which is either CDPI₃ (C), CDPI₃ (N) andCDPI₄-(SO₃ ⁻)₂. The parameters were determined at PCR buffer, where theoligonucleotide concentration was 5×10⁻⁷M. The sequences below werehybridized to matched complement of ODN # 1. The relative free energydifference (ΔΔG°) between match and mismatch at positions 5, 6, 7 and 8were calculated at 50° C. and plotted in FIG. 2. The bold C base in thesequences indicates the position of the C/A mismatch.

SEQUENCES (SEQ ID NOS:2-6) #1 GGTTTTAATG-X″ #0 GGTTTCAATG-X″ #2GGTTCTAATG-X″ #3 GGTCTTAATG-X″ #4 GGCTTTAATG-X″

As shown in FIG. 2, the CDPI₄-(SO₃ ⁻)₂ (i.e., DPI₄-(SO₃ ⁻)₂) conjugateshowed comparable mismatch discrimination than that of the other twoCDPI₃ (i.e., DPI₃) conjugates. In addition, improved mismatchdiscrimination was observed when a mismatch is placed in the minorgroove binding area in the duplex. Thus, the CDPI₄-(SO₃ ⁻)₂ conjugateextends the useful binding area for this purpose.

All publications and patent applications cited in this specification areincorporated herein by reference in their entireties. The foregoingdiscussion of the invention has been presented for purposes ofillustration and description. The foregoing is not intended to limit theinvention to the foam or forms disclosed herein. Although thedescription of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1. A negatively charged minor groove binding compound comprising abinding moiety that binds preferentially into a minor groove of a doublestranded oligonucleotide, wherein said binding moiety comprises: atleast one aryl moiety, and a plurality of acidic moieties capable ofionizing under physiological conditions, wherein each acidic moiety iscovalently attached to a phenyl moiety of said at least one aryl moietyor a heteroatom of a heteroaryl portion of said at least one arylmoiety, wherein said acidic moiety optionally comprises an acidic moietylinker.
 2. The negatively charged minor groove binding compoundaccording to claim 1, wherein said aryl moiety is selected from thegroup consisting of phenyl, a heteroaryl, a fused phenyl-heteroaryl, afused heteroaryl-phenyl-heterocyclyl and a combination thereof.
 3. Thenegatively charged minor groove binding compound according to claim 1,wherein said acid moiety has pKa of about 6 or less.
 4. The negativelycharged minor groove binding compound according to claim 1, wherein saidbinding moiety comprises a plurality of aryl moieties.
 5. The negativelycharged minor groove binding compound according to claim 4, wherein saidbinding moiety comprises at least three aryl moieties.
 6. The negativelycharged minor groove binding compound according to claim 4, wherein eachof said aryl moiety is independently selected from the group consistingof indole, benzofuran, pyrroloindole, hydropyrroloindole, phenyl,pyrrole, benzimidazole, imidazole, pyridine,6-phenylimidazo[4,5-b]pyridine, furan, thiazole and oxazole.
 7. Thenegatively charged minor groove binding compound according to claim 6 ofthe formula:

wherein n is an integer from 2 to 10; R³ is selected from the groupconsisting of: (a) alkoxy, (b) aryloxy, (c) R^(a)—O-L³-NR^(b)—, whereR^(a) is hydrogen or a hydroxyl protecting group; R^(b) is hydrogen,alkyl, cycloalkyl or a nitrogen protecting group, and L³ is a linkercomprising a chain of from 3 to 20 atoms selected from the groupconsisting of C, N, O, S, P and combinations thereof, and (d) a moietyof the formula:

each R⁴ is independently hydrogen, alkyl or said acidic moiety,optionally comprising said acidic moiety linker; each X³ isindependently selected from the group consisting of: (a) hydrogen, (b)alkyl, (c) alkoxy, (d) halide, (e) cyano, (f) nitro, (g)—NR^(b1)—C(═O)—R^(e), where R^(b1) is hydrogen, alkyl, cycloalkyl or anitrogen protecting group and R^(e) is hydrogen, alkyl or cycloalkyl,and (h) said acidic moiety, optionally comprising said acidic moietylinker; each R⁵ is independently selected from the group consisting of(a) hydrogen, (b) alkyl, (c) alkoxy, (d) cycloalkyl, (e) halide, (d)cyano, (g) nitro, (h) —[X⁴]_(m1)—C(═O)—[O]_(m2)—R⁸, where each m1 and m2is independently 0 or 1, X⁴ is O or NR^(b1), where R^(b1)is hydrogen,alkyl, cycloalkyl or a nitrogen protecting group, and R⁸ is hydrogen,alkyl or cycloalkyl, provided when m2 is 1, R⁸ is alkyl or cycloalkyl,(i) —C(═O)—NR^(e)R^(f), where each of R^(e) and R^(f) is independentlyhydrogen, alkyl, cycloalkyl or a nitrogen protecting group, and (j) saidacidic moiety, optionally comprising said acidic moiety linker; each ofR⁶ and R⁷ is independently selected from the group consisting of: (a)hydrogen, (b) alkyl, (c) cycloalkyl, (d) -L^(x)Z^(x), where L^(x) is alinker comprising from 3 to 20 atoms selected from the group consistingof C, N, O, S, P and combinations thereof, and Z^(x) is selected fromthe group consisting of hydrogen, a protecting group and a solidsupport, (e) a moiety of the formula—(Z¹)_(j)—C(═O)—(R¹⁰)_(k)—[C(═O)]_(l)—R¹¹, where each of j, k and l isindependently 0 or 1; each Z¹ is independently selected from the groupconsisting of O, NR¹² and alkylene; each R¹⁰ is independently selectedfrom the group consisting of alkylene and cycloalkylene; each R¹¹ isindependently selected from the group consisting of alkyl, alkoxy,aryloxy, —NR¹³R¹⁴, —NR¹⁵—NR¹⁶R¹⁷, hydroxyalkyl and thioalkyl; and eachof R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ is independently selected from thegroup consisting of hydrogen, alkyl, cycloalkyl and a nitrogenprotecting group; and (f) said acidic moiety, optionally comprising saidacidic moiety linker; provided at least one of X³, R⁴, R⁶ and R⁷ is saidacidic moiety optionally comprising said acidic moiety linker.
 8. Thenegatively charged minor groove binding compound according to claim 7,wherein n is 2 to
 8. 9. The negatively charged minor groove bindingcompound according to claim 6 of the formula:

wherein W¹ is N or CR^(x30), where R^(x30) is hydrogen, alkyl, orhydroxy; W² is NR¹⁹, S or O; p is an integer from 2 to 12; each R¹⁹ isindependently selected from the group consisting of: (a) hydrogen, (b)alkyl, (c) a nitrogen protecting group, and (d) said acidic moietyoptionally comprising an acidic moiety linker; each of R¹⁸ and R²⁰ isindependently selected from the group consisting of: (a) hydrogen, (b)alkyl, (c) cycloalkyl, (d) said acidic moiety; and (e)—(Z¹)_(j)—C(═O)—(R¹⁰)_(k)—[C(═O)]_(l)—R¹¹ , where each of j, k and l isindependently 0 or 1; each Z¹ is independently selected from the groupconsisting of O, NR¹² and alkylene; each R¹⁰ is independently selectedfrom the group consisting of alkylene and cycloalkylene; each R¹¹ isindependently selected from the group consisting of alkyl, alkoxy,aryloxy, —NR¹³R¹⁴, —NR¹⁵—NR¹⁶R¹⁷, hydroxyalkyl and thioalkyl; and eachR¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ is independently selected from thegroup consisting of hydrogen, alkyl, cycloalkyl and a nitrogenprotecting group; provided at least one of R¹⁸, R¹⁹, or R²⁰ is saidacidic moiety, optionally comprising said acidic moiety linker.
 10. Thenegatively charged minor groove binding compound according to claim 9 ofthe formula:

wherein p, R¹⁸, R¹⁹ and R²⁰ are those defined in claim
 9. 11. Thenegatively charged minor groove binding compound according to claim 6 ofthe formula:

wherein R²¹ is an optionally substituted aryl-heterocyclyl; each of R²²and R²⁴ is independently selected from the group consisting of hydrogen,alkyl, and a nitrogen protecting group; Ar¹ is optionally substitutedaryl moiety; and R²³ is selected from the group consisting of hydrogenand said acidic moiety, optionally comprising said acidic moiety linker;provided when R²³ is hydrogen at least one of Ar¹ or R²¹ is substitutedwith said acidic moiety, optionally comprising said acidic moietylinker.
 12. The negatively charged minor groove binding compoundaccording to claim 11, wherein R²² and R²⁴ are hydrogen.
 13. Thenegatively charged minor groove binding compound according to claim 12,wherein R²¹ is of the formula:

wherein R²⁶ is selected from the group consisting of hydrogen, alkyl,and a nitrogen protecting group; R²⁷ is selected from the groupconsisting of: (a) hydrogen, (b) alkyl, (c) cycloalkyl, (d) said acidicmoiety, optionally comprising said acidic moiety linker, (e)—(Z¹)_(j)—C(═O)—(R¹⁰)_(k)—[C(═O)]_(l)—R¹¹ , where each of j, k and l isindependently 0 or 1; each Z¹ is independently selected from the groupconsisting of O, NR¹² and alkylene; each R¹⁰ is independently selectedfrom the group consisting of alkylene and cycloalkylene; each R¹¹ isindependently selected from the group consisting of alkyl, alkoxy,aryloxy, —NR¹³R¹⁴, —NR¹⁵—NR¹⁶R¹⁷, hydroxyalkyl and thioalkyl; and eachR¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ is independently selected from thegroup consisting of hydrogen, alkyl, cycloalkyl and a nitrogenprotecting group; and (f) -L^(x)Z^(x), where L^(x) is a linkercomprising from 3 to 20 atoms selected from the group consisting of C,N, O, S, P and combinations thereof, and Z^(x) is selected from thegroup consisting of hydrogen, a protecting group and a solid support,R²⁸ is selected from the group consisting of hydrogen and said acidicmoiety optionally comprising said acidic moiety linker.
 14. Thenegatively charged minor groove binding compound according to claim 12,wherein Ar¹ is selected from the group consisting of:

wherein each of R²⁹, R³⁰ and R³² is independently selected from thegroup consisting of hydrogen and said acidic moiety, optionallycomprising said acidic moiety linker; R³⁴ is selected from the groupconsisting of: (a) hydrogen, (b) alkyl, (c) cycloalkyl, (d) said acidicmoiety, optionally comprising said acidic moiety linker, (e)—(Z¹)_(j)—C(═O)—(R¹⁰)_(k)—[C(═O)]_(l)—R¹¹, where each of j, k and l isindependently 0 or 1; each Z¹ is independently selected from the groupconsisting of O, NR¹² and alkylene; each R¹⁰ is independently selectedfrom the group consisting of alkylene and cycloalkylene; each R¹¹ isindependently selected from the group consisting of alkyl, alkoxy,aryloxy, —NR¹³R¹⁴, —NR¹⁵—NR¹⁶R¹⁷, hydroxyalkyl and thioalkyl; and eachof R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ is independently selected from thegroup consisting of hydrogen, alkyl, cylcoalkyl, and a nitrogenprotecting group; and (f) -L^(x)Z^(x), where L^(x) is a linkercomprising from 3 to 20 atoms selected from the group consisting of C,N, O, S, P and combinations thereof, and Z^(x) is selected from thegroup consisting of hydrogen, a protecting group or a solid support;each of R³¹ and R³³ is independently selected from the group consistingof hydrogen, alkyl, a nitrogen protecting group and said acidic moietyoptionally comprising an acidic moiety linker; each of R⁴⁰, R⁴³ and R⁴⁴is independently selected from the group consisting of hydrogen, alkyl,and a moiety of the formula -L^(x)Z^(x),—(Z¹)_(j)—C(═O)—(R¹⁰)_(k)—[C(═O)]_(l)—R¹¹; each of R⁴¹ and R⁴² isindependently selected from the group consisting of hydrogen, alkyl, andsaid acidic moiety which optionally comprises said acidic moiety linker;and R⁴⁵ is selected from the group consisting of hydrogen, alkyl,alkoxy, cycloalkyl, halide, cyano, nitro, and a moiety of the formula—[X⁴]_(m1)—C(═O)—[O]_(m2)—R⁸, wherein each of m1 and m2 is independently0 or 1, X⁴ is O or NR^(b1), where R_(b1) is hydrogen, alkyl, cycloalkylor a nitrogen protecting group, and R⁸ is hydrogen, alkyl or cycloalkyl,provided when m2 is 1, R⁸ is alkyl or cycloalkyl.
 15. The negativelycharged minor groove binding compound according to claim 1 comprising atleast three of said acidic moieties.
 16. The negatively charged minorgroove binding compound according to claim 1, wherein each of saidacidic moiety is independently selected from the group consisting of:(i) —(O)_(d)S(O)_(e)OH, wherein d is 0 or 1 and e is 1 or 2, and (ii)—(O)_(f)P(O)_(g)(OR^(a1))_(h)(OH)_(i), wherein each R^(a1) isindependently selected from the group consisting of alkyl, aralkyl andaryl; f is 0 or 1; each of g and h is independently 0, 1, or 2, and i is1, 2 or 3, provided the sum of g+h+i is 2 or 3; (iii) —CO₂H; and (iv)salts thereof.
 17. The negatively charged minor groove binding compoundaccording to claim 16 further comprising said acidic moiety linker,wherein said acidic moiety linker comprises from 1 to about 20 atomsselected from the group consisting of C, N, O, S, P and combinationsthereof.
 18. The negatively charged minor groove binding compoundaccording to claim 17, wherein the combination of said acidic moiety andsaid acidic moiety linker is of the formula:—(X¹)_(a)—[C(═O)]_(b)—(R¹)_(c)X² wherein each of a, b and c isindependently 0 or 1, provided at least one of b and c is 1; each X¹ isindependently selected from the group consisting of: (i) O, (ii) NR²,where each R² is independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, and a nitrogen atom protecting group, and(iii) alkylene; each R¹ is independently selected from the groupconsisting of alkylene, cycloalkylene, arylene and a combinationthereof; and X² is said acid moiety.
 19. The negatively charged minorgroove binding compound according to claim 16, wherein X² is selectedfrom the group consisting of —SO₂OH, —OPO₂(OH), —CO₂H, and saltsthereof.
 20. The negatively charged minor groove binding compoundaccording to claim 1, wherein said negatively charged minor groovebinding compound is covalently attached to a solid support.
 21. Thenegatively charged minor groove binding compound according to claim 20,wherein said negatively charged minor groove binding compound isattached to said solid support through a solid support linker.